Acute kidney injury (AKI) or acute renal failure (ARF) is a sudden clinical syndrome of kidney failure or damage that happens within a few hours or days [1
]. AKI is manifested by an increase in serum creatinine and a reduction in urine output [1
], and it is caused by multiple factors, including toxic, ischemic, and immunologic insults, either individually or combined [3
]. The incidence and mortality of AKI are highly variable from 0.9% to 20% and from 25% to 80%, respectively, depending on the cohorts [4
]. Since current management strategies for AKI are limited to conservative treatments and waiting for recovery [6
], a substantial number of unmet medical needs persist for AKI treatment.
Mesenchymal stem/stromal cells (MSCs) have been applied to reduce the damage caused by renal injuries in preclinical studies [7
]. In addition, mounting evidence has demonstrated that exosomes derived from various MSCs (MSC-exosomes) isolated from different tissue sources, such as bone marrow, adipose tissue, umbilical cord tissue, and umbilical cord blood, have therapeutic potential against kidney diseases including AKI and chronic kidney disease (CKD), in multiple animal models [7
]. Exosomes are nano-sized, lipid-bilayered extracellular vesicles (EVs) that are shed by the fusion of multivesicular bodies (MVBs) with the plasma membrane, and they mediate mainly the paracrine effects of stem-cell therapy [10
]. Interestingly, most of the animal studies into the therapeutic effects of MSC-exosomes in kidney injuries have been performed with exosomes isolated almost exclusively by ultracentrifugation [8
]. The limitations of ultracentrifugation for exosome isolation include the production of exosomes with co-precipitated contaminants such as protein aggregates, the loss of exosome function because of their aggregation or distortion during the isolation process, and functional inhibition of exosomes by the media used in density gradient ultracentrifugation [12
]. Large-scale isolation of single-batch exosomes by ultracentrifugation is also restricted because of the limited instrumental capacity. This raises another problem as new batch of exosomes have to be produced for every experiment. As strict batch-to-batch consistency must be guaranteed for all biological products, ultracentrifugation-based isolation method may not be the optimal method to isolate exosomes for the development of therapeutics.
Many technical challenges need to be overcome in the development of exosome-based therapeutics [13
]. The large-scale production of high-quality exosomes is the most important factor in their therapeutic application. Among the various isolation methods, tangential flow filtration (TFF) has been proposed as the ideal method for industrial-scale manufacturing of exosomes [12
]. The TFF systems available for good manufacturing practice (GMP) are already in use and provide validated processes and GMP documents [15
]. In fact, TFF was first introduced in 2010 for the isolation of exosomes according to size [16
] and was gradually employed for exosome isolation or concentration in various experimental settings [17
]. More importantly, recent studies demonstrated the superior yield and activity of exosomes isolated by TFF compared with those isolated by ultracentrifugation [26
]. The high-purity isolation of exosomes is achievable with further diafiltration using TFF with appropriate pore sizes and parameters, including transmembrane pressure, flow rate, and the diafiltration factor [36
]. However, sophisticated optimizations of TFF might be required to preserve the surface-associated proteins which are important functional components of exosomes [38
]. Interestingly, no studies have used exosomes isolated using TFF for AKI application in animal models so far. Here, we describe the reproducible large-scale production and characterization of exosomes derived from human adipose tissue-derived MSCs (ASC-exosomes) using TFF and the life-saving efficacy of ASC-exosomes in a lethal model of AKI induced by cisplatin in the rat.
Until now, there has been no approved therapeutic intervention available for AKI [48
]. Various toxic or ischemic insults lead to tubular injury in AKI through multiple pathways, including microvascular dysfunction, oxidative stress, inflammation, immune dysregulation, cell death, and/or senescence [49
]. Numerous potential monotherapies targeting individual pathways are under clinical development [48
]; however, the targeting of multiple pathways is preferable to alleviate or inhibit the progression of complex diseases [50
Recently, kidney cells have been revealed to be capable of regenerating and repairing themselves throughout their lifetimes, which is in contrast to the traditional notion of the kidney as a static organ with limited cellular turnover and regenerative capacity [4
]. In fact, recovery from AKI depends on the regenerative capacity of renal tubules [4
]; the failure to replace injured tubular epithelial cells during recovery may lead to fibrosis and CKD [52
]. In this context, new therapeutics aimed at providing regenerative potential in addition to targeting multiple pathways might be the most promising form of AKI treatment.
It is now widely accepted that MSC-exosomes are the next-generation of regenerative therapeutics capable of targeting multiple pathways with regenerative function and overcoming the limitations of cell-based therapeutics [14
]. Several studies have already demonstrated the therapeutic effects of MSC-exosomes from different cell sources, including bone marrow, Wharton’s Jelly, and umbilical cords, in a diversity of AKI animal models [7
]. For instance, injection of human umbilical cord-MSC (UC-MSC)-derived exosomes reduced apoptosis and necrosis of proximal kidney tubules by ameliorating oxidative stress in a cisplatin-induced rat AKI model. In vitro, UC-MSC-exosomes induced proliferation of a renal tubular epithelial cell line and suppressed expression of caspase 3 and the authors show that these effects were mediated by activation of extracellular-signal-regulated kinase (ERK) 1/2 pathway [55
]. Another study by Zhang et al. shows that human Wharton’s jelly-MSC-derived extracellular vesicles protected kidney function of rats in ischemia-reperfusion injury (IRI) model by reducing oxidative stress via activation of nuclear factor erythroid 2-related factor 2 (NRF2) [56
]. In fact, NRF2 was reported as a cargo protein of ASC-exosomes which reduced the high glucose-induced premature senescence of endothelial progenitor cells [57
]. In addition, antioxidant enzymes peroxiredoxin (PRDX) 1, 4, and 6 have been found in the proteome of ASC-exosomes by our group [34
]. These results suggest that the anti-oxidative activity of ASC-exosomes might contribute the protection of life from lethal renal injury. Further study will be needed to decipher the molecular mechanism of this with GMP-grade ASC-exosomes for clinical translation. As observed in other disease models, various miRNA cargoes, such as miR-30, miR-199a-5p, miR-486-5p in MSC-exosomes also contribute to the protective and renal-regenerative effects in AKI models as well [58
]. However, the clinical translation of these studies is limited by available exosome-isolation methods; ultracentrifugation-based isolation methods were applied in all of the 31 reported studies on preclinical rodent models [9
]. Ultracentrifugation is the most widely used method for isolating exosomes from the MSC-CM [61
]. However, TFF-based methods are recognized as the most suitable methods for the manufacture of GMP-grade exosomes from large volumes of CM, with comparable high yield and purity as size exclusion chromatography-based methods [12
In the present study, we demonstrated the stable production of ASC-exosomes from cryopreserved ASCs. ASCs could be expanded up to passage 7 without loss of specific characteristics. The characteristics of ASCs were also maintained after incubation with serum-free media to obtain the CM. Using this process, several liters of CM could be obtained and further processed to isolate ASC-exosomes with a TFF-based method. Analysis of multiple batches of isolated ASC-exosomes demonstrated (1) the presence of stable characteristics, including size and surface markers; (2) the efficient removal of the cellular waste product (ammonium ion) and process impurity (BSA); and (3) the reproducibility and purity of the ASC-exosomes. Proteomic and lipidomic profiling analyzes resulted in comparable profiles in three batches of ASC-exosomes. More importantly, multiplex surface-marker profiling analysis successfully demonstrated a high degree of consistency in the levels of 37 surface-marker proteins in 11 different batches of ASC-exosomes. Overall, the established TFF isolation method allows for the reproducible production of exosomes with stable characteristics from large volume of culture media.
Previously, ASC-exosomes, isolated via ultracentrifugation, were reported to protect rat kidneys from acute ischemia-reperfusion injury [6
]. However, the therapeutic effects of exosomes might vary between isolation methods because the integrity of the isolated exosomes and the molecular composition of their cargo and/or surface-associated molecules differ among the isolation methods. Thus, ASC-exosomes isolated via TFF may show different cargo profiles from ASC-exosomes isolated via ultracentrifugation and different mode of actions in alleviating AKI.
The present study successfully evaluated, for the first time, the life-saving efficacy of ASC-exosomes isolated by TFF in a lethal model of AKI induced by cisplatin-challenge in rats. As this study mainly focused on the manufacturing aspect of ASC-exosomes, ASC-exosomes were applied in an extreme, lethal model of AKI to observe their protective and life-saving efficacies. To further delineate the underlying molecular mechanisms, the effects of ASC-exosomes must be tested in AKI model induced by sub-lethal dose of cisplatin and additional factors, such as renal histology, infiltration of immune cells and signaling pathways relevant to cell death, must be analyzed. Although further studies are needed to decipher the underlining molecular mechanisms, the isolation of ASC-exosomes by TFF might provide a scalable GMP process that facilitates the development of exosome-based therapeutics in the near future (Figure 7
4. Materials and Methods
4.1. Kits and Reagents
The reagents used in this study were purchased from the following sources: high glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, Dulbecco’s phosphate buffered saline (DPBS), Trypsin-EDTA, L-glutamine, sodium pyruvate, and Exosome-Human CD81 Flow Detection Reagent from Thermo Fisher Scientific (Carlsbad, CA, USA); differentiation medium for adipogenic, osteogenic, and chondrogenic lineage from Cefo Co., Ltd.(Seoul, Korea) or Thermo Fisher Scientific (Carlsbad, CA, USA); normal IgG, Phycoerythrin (PE)-conjugated antibodies for CD29, CD90, CD105, CD31, CD45, HLA-DR, CD9, CD63, and CD81 from BD Biosciences (San Jose, CA, USA); Pyrogent™ Gel Clot Limulus Amebocyte Lysate (LAL) assay for the bacterial endotoxin test from Lonza (Morristown, NJ, USA); e-Myco™ Mycoplasma PCR Detection Kit from iNtRON Biotechnology (Seongnam-si, Gyeonggi-do, Korea); ELISA for bovine serum albumin (BSA) from Shibayagi (Gunma, Japan); ELISA for calnexin from LSBio (Seattle, WA, USA); ELISA for cytochrome C from Abcam (Cambridge, UK) or R&D Systems (Minneapolis, MN, USA); detection reagents for ammonium ions from Roche Diagnostics (Mannheim, Germany); MACSPlex Exosome kit (human) from Miltenyi Biotec (Bergish Gladbach, Germany); 500-kDa molecular weight cut-off filter membrane cartridge from GE Healthcare (Chicago, IL, USA) or Pall (Port Washington, New York, USA); and cisplatin from Sigma Aldrich (St. Louis, MO, USA).
4.2. Culture and Characterization of ASCs
A human ASC cryostock of passage 4 was prepared as described previously [34
] and stored in liquid nitrogen. Frozen cells were thawed at 37 °C, plated at a density of 3000 cells/cm2
, and cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin at 37 °C and 5% CO2
. Cultured ASCs were sub-cultured before the cultures reached confluence. The viability and size of the cells were monitored by an automated cell counter with trypan blue staining [63
]. ASCs were characterized for surface-marker expression and trilineage differentiation potentials according to the criteria described by the International Society of Cellular Therapy [64
4.3. Isolation and Characterization of ASC-Exosomes
To obtain ASC-CM, a vial of ASC stock was thawed and sub-cultured with gradually increasing culture scales in a T175 flask and a 1- or 2-layered Cell Factory Systems (Thermo Fisher Scientific; Carlsbad, CA, USA) before passage 7 at 37 °C and 5% CO2. At passage 7, the ASCs were plated at a density of 6000 cells/cm2 in 10-layered Cell Factory Systems and cultured up to 90% confluency in DMEM containing 10% FBS at 37 °C and 5% CO2. The ASCs were washed three times with DPBS to remove FBS and supplemented with serum- and phenol-red-free DMEM containing 1% L-glutamine (200 mM) and 1% sodium pyruvate (100 mM). The cells were further incubated for 24 h at 37 °C and 5% CO2 before the CM were collected.
ASC-exosomes (ASCE™, ASCE is the proprietary trademark of ExoCoBio) were isolated from ASC-CM using the TFF-based ExoSCRT™ technology as previously described [23
]. Briefly, to remove larger non-exosomal particles, including cells, cell debris, microvesicles, and apoptotic bodies, the ASC-CM were filtered through a 0.22-μm polyethersulfone membrane filter (Merck Millipore, Billerica, MA, USA) and then concentrated by TFF with a 500 kDa molecular weight cut-off filter. The concentrated ASC-CM was further diafiltrated with appropriate volumes of PBS to remove non-exosomal proteins, nutrients, and cellular waste products such as lactate and ammonia. Isolated ASC-exosomes were stored at –80 °C as small aliquots in sterile polypropylene tubes. The frozen ASC-exosomes were stored at 4 °C until completely thawed before further use.
Characterization of the ASC-exosomes was performed according to the Minimal Information for Studies of Extracellular Vesicels 2018 (MISEV2018) recommended by the International Society for Extracellular Vesicles [65
]. NTA was performed with a NanoSight NS300 (Malvern Panalytical, Amesbury, UK) as described previously [23
]. TEM analysis, FCM analysis, and protein quantification were also conducted as described previously [23
]. ELISAs for calnexin, cytochrome C, and BSA were performed according to the manufacturer’s recommendations. Measurements of ammonium ions were performed according to the manufacturer’s instructions. Proteomic and lipidomic analyses of ASC-exosomes were conducted as described previously [34
4.4. Animal Study
The animal study was approved by the Knotus Institutional Animal Care and Use Committee (IACUC; approval number 17-KE-315, November 10, 2017) and performed according to the Animal Experimentation Policy of Knotus Co., Ltd. (Incheon, Korea). Six week-old male SD rats were obtained from Orient Bio (Seongnam, Gyonggi-do, Korea) and kept under controlled environmental conditions (temperature: 23 ± 3 °C; relative humidity: 55 ± 15%; ventilation: 10–20 air changes/hr; and luminous intensity: 150–300 Lux) with a 12-h light-dark cycle in the experimental animal facility at Knotus. AKI was induced by IP injection of 10 mg/kg cisplatin that was dissolved in saline as described previously [66
]. At 8 h after cisplatin injection, the vehicle control or the increased amounts of ASC-exosomes were IV administered at a flow rate of 1 mL/min through the tail vein. For repeated injection, ASC-exosomes were further administered 48 h after the first injection. The amounts of ASC-exosomes were 3.09 × 109
and 10.3 × 109
particles/head (corresponding to 21 and 70 μg of protein, respectively). The animals’ body weights and survival were monitored daily. Blood samples were collected on day 0 (before cisplatin injection), 2, 4, 6, 8, and 12, and analyzed for blood urea nitrogen (BUN) and creatinine (CRE) with the 7180 Clinical Analyzer (Hitachi High-Technologies, Tokyo, Japan).
4.5. Statistical Analysis
Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) was used to analyze data. Comparisons among different groups were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison of multiple means. All values are expressed as mean ± SEM. p-values of *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.