Unlike the standard cell culture environment with regular oxygen levels (21%), transplanted MSCs face a hypoxic microenvironment in injured tissues. Culturing MSCs under hypoxic conditions enhance the function of MSCs by increasing proliferation, survival, homing, differentiation and paracrine activities (30-33), which may enhance their therapeutic potential in treatment of AKI.

Hypoxic preconditioning of MSCs has shown promising results in the treatment of AKI. Various oxygen concentrations have been tested for preconditioning MSCs, with different sources of MSCs and animal models. MSCs preconditioned under low oxygen conditions (1% O₂, 5% O₂ and CoCl₂) contribute markedly to kidney function restoration; 1% O₂-preconditioned human adipose-derived MSCs (hADMSCs) in rats with ischemia-reperfusion injury (IRI) showed improvements in apoptosis, enhanced anti-oxidative capacity and increased vascularization, leading to overall renal function improvement (34). Furthermore, 1% O₂-preconditioned hADMSCs showed enhanced immunomodulatory effects, improved angiogenesis and reduced oxidative stress, leading to improved renal preservation (35). Additionally, 1% O₂-preconditioned human bone marrow MSCs led to a marked renal function restoration in IRI rat models (36).

Notably, higher oxygen concentrations, such as 5% O₂, also provided beneficial effects. A study reported that hypoxic preconditioning of human umbilical cord MSCs (hUCMSCs) with 5% O₂ in a gentamicin-induced acute renal failure rat model led to increased expression of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), integrins and stromal-derived factor-1, all of which ameliorated renal function (37). Moreover, a study showed that 5% O₂-preconditioned AD-MSCs in mice with IRI demonstrated an increase in EVs particle number and protein concentrations, which reduced oxidative reactions and protected kidney function (38). Similarly, rat BMSCs exposed to CoCl₂, which mimics the hypoxic environment, showed enhanced hypoxia-inducible factor 1 (HIF-1) and C-X-C chemokine receptor type 4 (CXCR4) expression, improved migration and prolonged retention, contributing to improved kidney preservation in IRI (39).

These findings highlight that hypoxic preconditioning is a potent strategy for optimizing MSCs-based therapies in AKI. The enhancement of key pathways such as angiogenesis, anti-oxidative mechanisms and immunomodulatory effects, along with improved cellular retention and migration, makes hypoxia preconditioned MSCs a promising candidate for clinical applications in AKI treatment. However, it is important to note that prolonged or excessive exposure to hypoxia may have a negative impact on cellular metabolism and proliferation, leading to unstable cell function or inducing apoptosis. The present review summarized the role of hypoxia pretreated MSCs in the preclinical model of AKI (Table I) (34-41).

To identify effective strategies for optimizing MSCs, researchers have explored chemical preconditioning approaches. These agents are primarily characterized by strong antioxidant, anti-inflammatory and tissue-repair properties. The aim is to protect the cellular membrane structure, improve the microenvironment of MSCs and enhance their stability within this environment. Recent studies which reported on drugs and chemical preconditioned MSCs in AKI models were summarized in Table II (42-46).

CZ is a powerful muscle relaxant (47). As an FDA-approved drug, CZ has been shown to enhance the expression of anti-inflammatory cytokines, chemokines and critical immunoregulatory enzymes indoleamine 2,3-dioxygenase by promoting Forkhead box protein O3 phosphorylation. This process induces MSCs to adopt an anti-inflammatory phenotype, thereby strengthening their immunosuppressive capacity without increasing immunogenicity (44). In vitro, CZ markedly reduces T-cell activation and proliferation, substantially boosting the immunosuppressive function of hUCMSCs. In Thy1.1 antibody-induced AKI animal models, CZ-pretreated hUCMSCs effectively attenuated renal inflammation and decreased fibrinoid necrosis of the glomeruli, indicating a superior protective effect against kidney injury (44).

Ator has been shown to possess anti-apoptotic, antioxidant and anti-inflammatory properties (48). Ator improves the microenvironment not only by synergistically enhancing the inherent effects of BMSCs but also by introducing additional therapeutic benefits to BMSCs transplantation (49). Cai et al (45) proposed a combined therapeutic strategy for IRI in a rat model. Rats were administered Ator (lipitor; 5 mg/kg/day by gavage) starting 3 days before MSCs transplantation and continuing until sacrifice. Immediately following IRI, BMSCs were delivered via the left carotid artery. The results indicated that the Ator + MSCs combination therapy could synergistically promote post-ischemic renal functional and morphological recovery. These benefits may stem from the statin-mediated suppression of oxidative stress and inflammation in the injured kidney by inhibiting TLR4 signaling, thereby creating a more favorable environment for engrafted MSCs (45).

The erythropoietin receptor (EpoR) is not only expressed on hematopoietic cells but has also been reported in specific renal cell types, including tubular epithelial cells and mesangial cells (50). Growing evidence from animal studies indicates that erythropoietin (EPO) directly acts on these renal cells to mitigate AKI, a protective effect attributed to its antiapoptotic, antioxidative, anti-inflammatory properties and angiogenesis (51). DPO, a genetically modified EPO analog, exhibits a prolonged half-life and enhanced stability, enabling reduced dosing frequency while retaining biological activity comparable to native EPO (52). Altun et al (46), rats treated ischemia reperfusion injury (IRI)-induced AKI with either DPO, MSCs, or both (MSCs and DPO concomitantly). The results demonstrated that the combined administration of MSCs and DPO provided superior renoprotection and mitigated tissue damage, although the underlying mechanisms require further investigation.

Vitamin E, a fat-soluble vitamin with antioxidant properties, can reduce reactive oxygen species (ROS) and prevent cell death, thus benefiting AKI therapy pathways (53-55). The therapeutic effects of combining BMSCs with Vitamin E in a gentamicin-induced AKI model have been shown to involve promoting tubular cell proliferation, inhibiting apoptosis and improving both renal function and pathological abnormalities (42). Another study (43) confirmed that co-administration of UCMSCs and Vitamin E markedly suppressed renal inflammation by modulating inflammatory cytokines [interleukin (IL)-lβ, tumor necrosis factor (TNF)-α and IL-10] in the AKI microenvironment, thereby reducing kidney injury. Moreover, this combined treatment achieved more favorable outcomes than using UCMSCs or Vitamin E alone.

Research has shown that ADMSCs from patients with stage 5 CKD, pretreated with metformin, can slow down the aging of CKD-MSCs (56). Subsequently, the study showed that metformin pretreated ADMSCs markedly attenuate renal inflammation and fibrosis in a CKD mouse model induced by unilateral ureteral obstruction (UUO). This finding suggests that even MSCs derived from CKD patients, can be functionally optimized through agents such as metformin and developed as patient-derived, autologous MSC-based therapeutic products for CKD, offering a novel approach to personalized stem cell therapy (56). Hu et al (57) demonstrated that CA-pretreated BMSCs from mice could alleviate renal fibrosis by inhibiting the TGF-β1/TNF-α/TNFR1 pathway. However, there have been no published studies combining these two drugs with MSCs for the treatment of AKI and further research is needed.

Overall, multiple studies have confirmed that chemical drug preconditioning effectively enhances the antioxidative and anti-inflammatory abilities of MSCs. However, these drugs may have certain effects on the kidneys or other organs. Therefore, the potential side effects and long-term impacts of drug preconditioning need to be further evaluated through additional research.

MSCs typically exhibit low immunogenicity, thereby rarely triggering strong rejection responses in allogeneic transplantation (58,59). Moreover, research has demonstrated that the immunomodulatory and immunosuppressive capacities of MSCs can be further activated when exposed to inflammatory stimuli or residing within an inflammatory microenvironment (60-62). Numerous studies have shown that both exogenous and endogenous pro-inflammatory stimuli, such as interferon gamma (IFN-γ) (63), TNF-α (64,65), IL-17 (66), IL-1β (67), Toll-like receptor (TLR) (68) and TLR ligands (69), can activate or precondition MSCs, enhancing their secretion of immunoregulatory factors and their therapeutic potential. Current preconditioning studies targeting AKI have primarily focused on IFN-γ, IL-17 and IL-1β. While several reports indicate that TNF-α and TLR4 ligand-preconditioned MSCs can boost therapeutic efficacy in other disease models (65,70-72), there is relatively little published literature regarding the efficacy of TNF-α or TLR4-induced MSCs preconditioning in improving AKI outcomes. Additionally, the combined administration of MSCs with low doses of hormones has demonstrated significant renoprotective effects in various animal models (73,74). Biological compound preconditioning MSCs may offer novel insights into enhancing their therapeutic applications in AKI via hormones and cytokines. The recent findings were summarized in Table III (75-81).

IL-17A modulates MSCs immunoregulatory functions without affecting MHC expression (66,82,83), has been shown to enhance the therapeutic potential of BMSCs in mouse models of AKI (76). Mechanistically, IL-17A induces the expansion of regulatory T cells (Tregs), thereby reinforcing immunosuppression and repairing renal function. Conversely, the absence of Tregs diminishes T-cell suppression and negates the beneficial effects of IL-17A preconditioning (76).

IFN-γ has been employed to pretreat MSCs (63,84). IFN-γ-preconditioned BMSCs administered via the abdominal aorta in rat models of IRI and UUO exhibited marked reductions in immune cell infiltration and renal fibrosis (78). In mechanism, IFN-γ-preconditioned BMSCs upregulated PGE₂ related responses, which induced the polarization of immunosuppressive CD163⁺CD206⁺ macrophages in the local renal microenvironment. This immunomodulatory shift markedly attenuated inflammatory cell infiltration, restored immune homeostasis and ultimately protected against IRI-induced AKI (78).

IL-1β has been used to pretreat MSCs and has been shown to enhance MSCs immune regulation functions. Intravenous injection of IL-1β pretreated MSCs in hemorrhagic shock rats resulted in decreased plasma creatinine, urea nitrogen, cystatin C and kidney injury molecule kidney injury molecule 1 levels on renal tissue sections. In vitro experiments demonstrated that IL-1β-pretreated MSCs reduced systemic cytokines (IL-1α, IL-6 and IL-10) of monocytes and granulocytes, as well as CD80 and CD86, inhibiting activation of PD-1/PDL-1 axis. This suggests that IL-1β enhances MSCs efficiency by promoting its immunomodulatory activity (75).

Melatonin, a neurohormone primarily secreted by the pineal gland, plays a crucial role in regulating circadian rhythms (85-89). It is recognized as an effective free radical scavenger with protective effects on various dysfunctional organs, including the kidney (73,74). Given that inflammation and oxidative stress are key contributors to the impaired function of transplanted MSCs in disease environments, preconditioning MSCs with melatonin may prove beneficial (73,90,91). Animal studies in models of lung injury and cerebral and limb ischemia have demonstrated that melatonin preconditioning enhances the therapeutic outcomes of MSCs (92-96). Notably, melatonin not only exerts potent antioxidant effects by reducing free radical production, but also its receptors, MT1 and MT2, widely expressed on MSCs, may also modulate cell fate via receptor-dependent mechanisms (97-99). AKI caused by sepsis is a particularly severe subtype with high morbidity and mortality. Chen et al (77). compared melatonin-preconditioned ADMSCs with ADMSCs alone in a cecal ligation and puncture model, finding that the combined therapy produced superior anti-inflammatory, antioxidant, anti-apoptotic and anti-fibrotic responses, as well as lower serum creatinine levels and reduced renal damage on histological examination. In vitro, melatonin preconditioning markedly enhances ADMSCs proliferation and upregulates key survival and antioxidant proteins such as phosphorylated Erk1/2, Akt, SOD-1 and heme oxygenase-1 (HO-1) (77). The conditioned medium from these cells further promoted proliferation, migration and anti-apoptotic effects in cisplatin-exposed human renal epithelial cells (80). Moreover, Saberi et al (100) demonstrated that pretreatment of BMSCs with melatonin enhanced their homing to the injured kidney, reduced renal cell apoptosis, decreased the expression of TNF-α, α-SMA and TGF-β1 and increased E-cadherin levels in UUO. Ultimately, this approach improved the integrity of the tubular basement membrane, ameliorated tissue architecture and effectively mitigated renal fibrosis.

EPO is an endogenous hormone primarily secreted by the kidney that regulates red blood cell production (101). It stimulates the proliferation and differentiation of hematopoietic stem cells (HSCs) in the bone marrow through signaling via the EpoR (102). Notably, both BMSCs and HSCs express homologous EpoR. EPO has shown therapeutic potential in various conditions such as myocardial infarction (103), cerebral ischemia (104), chronic heart failure (105) and kidney injury (101,106) through its anti-inflammatory and antioxidant effects. Zhou et al (107). demonstrated that 48-h EPO pretreatment markedly enhances the proliferation rate, cytoskeletal reorganization, migration capacity and expression of the chemokine receptor CXCR4 in BMSCs, thereby improving their homing ability and cellular functionality in vitro. Subsequently, compared with untreated BMSCs, EPO-preconditioned BMSCs not only more effectively restored renal function but also markedly reduced interstitial lymphocyte infiltration, tubular swelling, necrosis and interstitial fibrosis in a cyclosporine A-induced nephrotoxicity rat model (107). Zhou et al (81) further reported that EPO pretreatment upregulated the expression of SIRT1 and the anti-apoptotic protein Bcl-2 while downregulating the pro-apoptotic protein p53 in BMSCs, thereby enhancing their survival rate and anti-apoptotic capacity. The authors observed that in a rat AKI model, GFP-labeled BMSCs infused after 24 h primarily accumulated in the lungs, whereas EPO pretreatment reduced pulmonary retention and increased their distribution to the injured kidneys. Moreover, AKI rats treated with EPO-preconditioned BMSCs exhibited markedly lower serum levels of IL-1β and TNF-α, along with elevated IL-10 levels and demonstrated more pronounced improvements in renal function (81). These findings indicate that EPO pretreatment not only enhances the survival and anti-apoptotic capabilities of BMSCs but also improves their homing and distribution in vivo, thereby markedly augmenting their therapeutic efficacy in AKI.

Relaxin is primarily secreted by the corpus luteum during pregnancy, with minor expression in the brain, heart and kidneys (108). It plays a key role in mediating adaptive hemodynamic changes, including increased cardiac output, enhanced renal blood flow and improved arterial compliance (109-113). Several investigations have explored relaxin preconditioning of MSCs (114,115). Although direct evidence of relaxin preconditioned MSCs protecting against AKI is currently lacking, combined therapy has been shown to mitigate fibrosis progression in a UUO model in normotensive mice (116,117). Badawi et al (117) developed a novel combinatorial approach for CKD patients using synthetic relaxin (Serelaxin) with BMSCs to restore and enhance endothelial progenitor cell populations. While BMSCs alone have demonstrated blood pressure reduction and anti-fibrotic effects comparable to perindopril, serelaxin monotherapy markedly decreases tubular injury and moderately reduces renal fibrosis (114). These findings suggest that further research is needed to fully elucidate the renoprotective potential of relaxin preconditioned MSCs in the setting of AKI.

In summarily, biological preconditioning strategies enhance the therapeutic effects in AKI by activating the immunomodulatory function of MSCs through cytokines or hormones. However, its main drawback is the potential risk of overstimulation, leading to unwanted side effects, such as excessive inflammation or immune modulation, which could complicate therapeutic use.

Conventionally, cell cultures are primarily performed on flat, two-dimensional substrates. Nevertheless, in a two-dimensional setup, the interplay among cells and between cells and the extracellular matrix (ECM) is not entirely consistent with that in vivo. In order to more closely mimic the in vivo microenvironment and improve cellular function as well as the physiological relevance of experimental data, MSCs 3D cell culture methods have been developed and applied in the study of AKI (Table IV) (118-125).

Hydrogels are regarded as one of the most promising options for MSCs 3D culture and delivery (126,127). Hydrogels, formed through chemical or physical crosslinking, are 3D porous polymer networks with high water content and tunable chemical and physical attributes (126,128). Owing to their substantial water content and porous structure, hydrogels facilitate the diffusion of nutrients and metabolites within the network (129). In this way, hydrogels can serve as an artificial ECM around the cells, providing the necessary conditions for cell-to-matrix and cell-to-cell interactions, thus influencing MSCs behavior and function (130). At present, hydrogels have been explored to replicate the native microenvironment of cells in vivo and they are categorized by source or fabrication approach into natural hydrogels, synthetic hydrogels and hybrid hydrogels (130). Notably, the effectiveness of hydrogels as scaffolds to support cellular growth and function has been confirmed in various preclinical AKI models (119,121,131).

Hydrogels made from natural materials have gained significant interest in recent years because of their excellent biocompatibility, biodegradability and eco-friendly nature (132). Common natural polymers utilized are agarose, alginate, chitosan, collagen, hyaluronic acid, gelatin and fibrin (133-136). These materials can be categorized as either polysaccharide-based or protein-based hydrogels (137).

Alginate, a linear hydrophilic polysaccharide from brown algae and specific bacteria, comprises β-d-mannuronic acid and α-l-guluronic acid (135). This biopolymer, soluble and cost-effective, with high biocompatibility and appropriate rheological properties, is widely used in 3D bioprinting (138). Before its bioink application, alginate was extensively researched in regenerative medicine and tissue engineering for MSCs culture and delivery (139-141). It further facilitates the regulated secretion of paracrine factors derived from MSCs (142,143). Nonetheless, the limited biodegradability and cell-adhesive characteristics of alginate restrict its potential uses (138,139). Efforts are ongoing to address these constraints. A recent study proposed that an alginate-hyaluronic acid hydrogel blend could be a promising option for AKI treatment (121).

Chitosan is a naturally occurring linear polysaccharide, is derived from crustacean exoskeletons and fungal cell walls (134,139). Beyond its structural resemblance to glycosaminoglycans in the ECM, it exhibits biocompatibility, biodegradability, antimicrobial properties, non-toxicity and affordability (144). These hydrogels demonstrate notable responsiveness to changes in pH and temperature (145), enhancing their adaptability for diverse applications. In vivo research has shown that chitosan hydrogels improve MSCs retention, viability and therapeutic efficacy in AKI (118,146). However, their weak mechanical properties and limited solubility in physiological environments restrict biomedical utility. Combining chitosan with peptides or alternative hydrogels offers a potential solution to these limitations (137,145).

Collagen is a fibrous structure, constitutes the predominant structural component in mammalian extracellular matrices (147). Owing to properties such as biodegradability, biocompatibility, elasticity and resemblance to natural tissue structures, collagen-based hydrogels are widely utilized as biomimetic scaffolds for 3D culture applications (139). Evidence indicates that scaffolds constructed from collagen effectively promote MSCs retention, proliferation, functional performance and phenotypic stability, consequently augmenting their therapeutic potential in treating AKI (122,139,148). A decellularized vascular matrix combined with collagen (co-gel) and MSCs were jointly transplanted into the kidneys of rats with IRI-induced AKI, the survival rate of MSCs and their paracrine effects in the injured kidneys were improved (122). More importantly, the co-gel markedly augmented the therapeutic efficacy of MSCs in AKI, including reducing cell death and tissue damage and improving angiogenesis and renal function (149). Duragen is an absorbable collagen-based artificial matrix that was used as a biological membrane to encapsulate MSCs transplanted into AKI mouse kidneys, preventing MSCs escape, prolonging their survival in vivo, improving renal function, ameliorating tubular lesions and reducing apoptosis. The authors also demonstrated in cell-based experiments that duragen-encapsulated MSCs protected renal cells from myoglobin-induced apoptosis. Thus, this confirms the potential of duragen-encapsulated MSCs as a new therapeutic strategy for AKI (150).

Gelatin, a biocompatible polypeptide obtained from bovine or porcine collagen, comprises 18 distinct amino acids (139,151) and possesses lower antigenicity relative to collagen (151). Due to its advantageous properties, such as affordability, commercial accessibility, water solubility, good adhesion and straightforward processing, gelatin has become widely adopted in biomedical applications (152). Research involving AKI models demonstrates that gelatin-based hydrogels combined with MSCs effectively promote MSC survival, thus facilitating tissue repair (119). However, challenges of gelatin hydrogels include low mechanical strength, quick enzymatic breakdown and poor thermal stability (153). Pure gelatin undergoes a sol-gel shift close to body temperature, allowing it to be injected as a thin liquid at 37°C, but it does not solidify into a stable hydrogel in vivo (154). Modifications are necessary to enhance the overall properties of natural gelatin.

Kidney ECM hydrogels have attracted significant interest as biomaterials for regenerative medicine (155). By selectively removing cellular components while preserving the native proteins, glycosaminoglycans and growth factors, these hydrogels maintain essential bioactive properties, biocompatibility and minimal immunogenic responses (156,157). Compared with hydrogels derived from individual ECM components, kidney ECM hydrogels uniquely preserve the complete biochemical profile of renal tissue and do not include proteins obtained from tumorigenic sources as does Matrigel (158). Kidney ECM hydrogels are currently being explored as injectable scaffolds to facilitate renal regeneration and tissue repair, showing promising outcomes (159). In a related investigation, embedding ADMSCs within kidney ECM hydrogels markedly enhanced cell retention and survival after transplantation into ischemic rat kidneys. Moreover, ECM hydrogels promoted growth factor secretion while reducing oxidative damage and apoptosis, suggesting therapeutic promise for AKI treatment (160). However, due to inherent variability associated with natural biomaterials, the characteristics of kidney ECM hydrogels and their specific influences on cellular behavior require further elucidation (158). Additional studies should further clarify these issues to provide deeper insights into AKI management.

HA is extensively found within mammalian tissues, including connective tissues, synovial fluid and vitreous humor (61,78). HA exhibits distinctive properties, including inherent bioactivity, excellent hydrophilicity, biodegradability and low cellular adhesiveness, making it a promising biomaterial candidate (145). These advantages make HA-based hydrogels increasingly suitable for numerous biomedical applications. As a crucial ECM constituent, HA markedly contributes to various biological processes, such as cellular proliferation, angiogenesis, embryogenesis, wound repair, stromal structuring and morphogenetic events (126). Implanting stem cells in HA hydrogels influences the release of cytokines/chemokines, counteracts pro-inflammatory mediators secreted by immune cells, thereby modulating immune responses and improving AKI (161). A previous investigation demonstrated that injecting MSCs encapsulated within HA hydrogels directly into kidneys subjected to IRI effectively attenuated ECM remodeling at one month post injury in mice (162). Notably, the highly thermoreversible nature of HA hydrogels provides favorable conditions for their use as injectable scaffolds for MSCs culture and delivery, or as implantable materials for soft and hard tissue repair and reconstruction (163). However, due to insufficient stability at body temperature and limited controlled-release capacity for bioactive molecules, chemical modifications and covalent crosslinking are required to improve the performance of HA hydrogels (164). Anyway, the aforementioned research proved that HA hydrogels have the potential to alleviate AKI.

Agarose (129), Matrigel (165) and fibrin (166)-based hydrogels have also been employed to culture and deliver MSCs and serve as viable scaffolds for MSCs cultivation, but research on their application in AKI remains limited and warrants further investigation.

Synthetic hydrogels are artificially engineered, 3D polymer networks that can absorb and retain large amounts of water, mimicking the properties of the natural ECM. They are widely used in biomedical applications such as tissue engineering, drug delivery and wound healing. Fu et al (119) discovered a novel therapeutic strategy for rhabdomyolysis. The authors used 3D modeling and printing to create an elastic sac that mimicked the kidney's dimensions and shape. This sac was applied as an outer coating for the kidney along with MSC-laden hydrogel (119). This innovative technology not only offers a versatile solution for organ restoration but also presents a promising option for AKI treatment. In another study, a new β-sheet supramolecular self-assembling peptide hydrogel was developed by combining insulin-like growth factor-1C (IGF-1C) with a D-assembling motif Nap-DFDFG, along with human placenta-derived mesenchymal stem cells for AKI. The results demonstrated that this hydrogel enhanced cell engraftment, ameliorated renal function, stimulated angiogenesis and reduced renal fibrosis in a mouse model of IRI induced AKI (125). Furthermore, an injectable nitric oxide (NO)-releasing hydrogel was created through physical crosslinking using short aromatic dipeptides. This hydrogel (Fmoc-diphenylalanine S-nitroso-N-acetylpenicillamine; Fmoc-FFSNAP) enabled localized and sustained NO release, leading to enhanced regeneration post-IRI and improved renal function (167). A recent study compared the therapeutic efficacy of Fmoc-FF + Fmoc-RGD-MSCs with free MSCs and Fmoc-FF + Fmoc-RGD-SNAP-MSCs in a renal IRI injury model. The hydrogel was combined with Wharton's jelly-derived MSCs and administered via intrarenal injection at three sites in IRI mice. Results demonstrated that the hydrogel treatment markedly enhanced renal function biomarkers, decreased reactive oxygen species production, improved histopathological changes and promoted the recovery and endothelial regeneration of the injured kidney (123).

Feng et al (118) demonstrated that a bioactive hydrogel, formed by immobilizing the C domain peptide of IGF-1C on chitosan, provided an artificial microenvironment for adipose-derived ADMSCs, which enhanced cell viability through paracrine effects and protected against IRI.

An alternative approach to 3D cell culture involves spheroid formation. This scaffold-free method enables MSCs and their ECM to self-assemble into tissue-like structures. Various techniques facilitate spheroid production, including liquid overlay, rotary culture, magnetic levitation, microparticle culture and the hanging drop method (168,169). Research suggests therapeutic applications for MSCs spheroids in kidney disorders. For example, Xu et al (124) demonstrated the enhanced therapeutic effects of human ADMSCs spheroids in AKI models. Compared with traditional monolayer cultures, these 3D aggregates showed increased secretion of ECM components (collagen I, fibronectin and laminin) along with superior antioxidative and anti-apoptotic capabilities.

Additionally, MSCs cultured with 3D spheroids exhibit enhanced secretion of paracrine cytokines such as VEGF and bFGF (angiogenic factors), EGF and HGF (anti-apoptotic factors), IGF (antioxidant) and TSG-6 (anti-inflammatory). These spheroids display greater in vivo survival and sustained paracrine effects, aligning with in vitro observations. In rat models of IRI-induced AKI, intrarenal injection of 3D spheroids provides superior renal protection compared to 2D-cultured cells, mitigating apoptosis, tissue injury and enhancing angiogenesis and functional recovery (124). Hybrid 3D spheroids composed of MSCs, vascular endothelial cells and podocytes can mimic glomerular microenvironments. Transplantation of these constructs into the renal cortex of hypertensive nephropathy mice improves kidney function, suggesting that engineered hybrid spheroids could markedly enhance podocyte replacement strategies and therapeutic outcomes (170).

3D culture of MSCs offers significant advantages in enhancing cell function and therapeutic effects, but it may also pose certain risks. First, the technical complexity and controllability of 3D culture systems are relatively low, which may lead to abnormalities in cell growth and differentiation. Second, hypoxia may occur in the center of the cells, affecting their survival and function. In addition, the therapeutic effects of 3D culture may vary due to inconsistencies in culture conditions and it may trigger immune rejection responses. Finally, the long-term effects and safety are not yet clear, particularly in clinical applications and further optimization and evaluation are needed.

To enhance MSCs therapeutic potential in AKI, intracellular engineering methods such as genetic and epigenetic modifications have been extensively studied. Genetic modification involves changing MSCs' DNA sequence to regulate the expression of important genes. For example, boosting HO-1 and VEGF, genes crucial for antioxidant defense and angiogenesis, has been found to notably enhance MSCs migration, vascular targeting, anti-inflammatory abilities and viability, ultimately improving renal healing (171). These protective impacts are mainly mediated through activating signaling pathways such as PI3K/Akt and MEK/ERK (172,173).

Gene modification currently used include viral vector-based systems such as lentiviruses, retroviruses, adenoviruses and adeno-associated viruses, as well as non-viral systems such as plasmid DNA, RNA and protein-RNA complexes (171,174). In AKI preclinical models, these approaches have been used to boost the regenerative and immunomodulatory capabilities of MSCs, enhancing their proliferation, influencing differentiation potential and enhancing their anti-inflammatory and antioxidative functions (Table V) (175-182). Despite encouraging outcomes, the clinical application of these methods is restricted. Genetic modification might unintentionally affect genomic regions outside the target, leading to potential risks of genotoxicity, cytotoxicity, or tumorigenesis (183). Furthermore, ethical and safety issues associated with gene editing technologies are impeding their broad clinical implementation. Therefore, although genetic manipulation is a potent tool for refining MSC-based therapies for AKI, further optimization and thorough safety assessment are crucial before clinical adoption.

The mechanisms by which preconditioning strategies enhance MSCs function can be briefly summarized as follows: Physical preconditioning (such as hypoxic culture) enhances MSCs function by promoting proliferation, survival, homing, differentiation and paracrine activity, thereby improving renal function recovery. Chemical preconditioning primarily enhances MSCs stability by protecting cell membrane structures and improving the MSCs microenvironment through antioxidant, anti-inflammatory and tissue repair properties. Biological preconditioning activates MSC's immunomodulatory functions through cytokines or hormones, further enhancing their therapeutic effects. In addition, genetic modification optimizes MSC therapy for AKI by altering gene expression to enhance MSCs migration, vascular targeting and anti-inflammatory abilities. Further mechanisms specific pathways are still being explored.

Although these strategies have their advantages, they also present some challenges. Physical preconditioning and 3D culture methods are relatively simple, but there may be variability in cell growth and differentiation consistency. Chemical and biological preconditioning methods are comparatively simple and have high reproducibility, but more research may be needed to evaluate their long-term effects and potential side effects. Genetic modification strategies can markedly enhance MSCs functionality, but their safety and ethical concerns still need further evaluation. A combination of these strategies may be the best approach for treating AKI in the future, but further research and optimization are still required.

To fully leverage the diverse therapeutic capabilities of MSCs, it is crucial to ensure the adequate delivery of cells to the site of injury, which is a fundamental requirement for the efficacy of MSC-based therapy. The selection of the administration route markedly influences therapeutic results, with no consensus on the most effective approach to date (148). Systemic and local injections are currently the main methods used in preclinical and clinical studies, while innovative strategies such as encapsulated delivery of biomaterials and pulsed focused ultrasound (pFUS) guidance are gaining attention (184) (Fig. 3).

Systemic administration included intransient (IV), intra-arterial (IA), intraperitoneal (IP), accounting for 74% of MSCs injected. IV is commonly used for systemic delivery of bone marrow MSCs because it is simple and minimally invasive, with tail vein injection being the most dominant mode. However, an important drawback is the 'lung first-pass effect', which causes MSCs to become trapped in the lungs (185,186). In addition, MSCs tend to accumulate in the liver after intravenous injection, thereby reducing their concentration to reach the kidneys (187). Although administering higher numbers of MSCs could mitigate this problem, it potentially elevates the likelihood of complications, including thrombotic events and pulmonary embolism (188-190). By contrast, IA has shown higher efficacy by bypassing lung filters, improving MSCs homing and increasing cell delivery to target tissues, despite the risk of cell embolism and higher invasiveness (191,192). Whether intraperitoneally administered MSCs can enter the systemic circulation has not been reported, although they have the ability to reach the renal parenchyma.

Topical administration accounts for ~40% of MSCs injection, including intra-renal, subcapsular, renal-arterial. The MSC sheet transplantation has emerged in recent years as a novel localized delivery strategy for MSCs. Like above topical administration, this approach enables direct and precise renal implantation, enhancing cellular engraftment and therapeutic efficacy while circumventing pulmonary entrapment and reducing pulmonary complication risks (193). Notably, MSC sheet transplantation uniquely maintains high local cell concentrations at target sites, thereby promoting renal tissue regeneration (193). Various topical administration methods have been evaluated in AKI models showing good results, with intra-renal injections being extensively studied for their ease of use. Other methods, such as hydrogel, microgel and biomaterials aforementioned, simulate the interaction between cells in vivo and improve the viability of MSCs. In addition, pFUS-guided delivery of MSCs was reported to improve microenvironment, up-regulate adhesion factors and guide MSCs homing and viability (184). Due to the small number of published reports, more studies are needed to confirm its safety and effectiveness.