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Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles | Stem Cell Research & Therapy

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Clinical-grade haMSC-EVs have suitable lot-to-lot consistency

The stability of the production process can usually be reflected by the lot-to-lot consistency of the products. The haMSC-EVs were isolated under the good manufacturing practice (GMP) standard, and CQCPs were determined during drug substance (DS) and drug product (DP) production processes to ensure product safety and quality. The DS production process used conditioned medium of haMSCs to isolate and purify the haMSC-EVs. The DS was stored in saline at high concentration (× 1010 particles/mL) after production. The DP production process used saline to dilute haMSC-EVs to 2–8 × 108 particles/3 mL/dose which was ready for use. Specifically, the QC evaluation included (1) safety tests of the cell culture supernatant, including endogenous virus, exogenous virus, and mycoplasma tests; (2) safety tests of the DS (including sterility, mycoplasma, and exogenous virus tests); particle analysis, protein concentration and marker profiling; and (3) safety tests and particle analysis of the DP. The haMSC-EVs used in the DS analysis met all the release criteria. The vesicles in the products showed a cup-shaped morphology in the TEM images, and a representative image is shown in Fig. 1A. The size distributions of 5 lots of drug substances analyzed by NTA are shown in Fig. 1B. The median sizes were between 100 and 150 nm. Based on the Western blot results, compared with the parental cells, the haMSC-EVs were enriched in CD9/63/81 and HSP70 and depleted of CANX (Fig. 1C). To further characterize haMSC-EVs, methodologies to test the marker expression ratios via nanoflow cytometry were developed using CD9, CD63 and CD81 antibodies and a PKH67 molecular probe. The minimum to maximum positive vesicle ratios of 5 batches were 59.4–73.2% for PKH67, 10.0–17.1% for CD9, 21.6–32.6% for CD63 and 17.9–26.3% for CD81. The relative standard deviations (RSDs) between the 5 lots were all less than 30% (9.06% for PHK67, 24.13% for CD9,17.06% for CD63 and 16.85% for CD81). The results suggest that the characteristics of our products are relatively stable. Multiple lots of haMSC-EVs were tested in the potency assay, and the TNF-α inhibition ratios were found to be greater than 30% (31.21% to 56.51% for the batches described above). Based on these existing quality studies, we updated our QC methodologies and summarized the corresponding criteria in Table 1. The quality of the products at the end points of the stability studies was evaluated based on these QC criteria.

Fig. 1
figure 1

EV characteristics were stable across multiple lots of haMSC-EVs. A Representative images of haMSC-EVs at different scales. B Size distribution of 5 lots of haMSC-EVs analyzed via NTA. C Representative images of the Western blot results for haMSC-EVs and parental cells. The haMSC-EVs were CD9-, CD63-, CD81-, and HSP70-positive and CANX-negative. Full-length blots are presented in Additional file 2: Fig. S1. D Marker expression ratios analyzed with nanoflow cytometry

Table 1 Quality Control of haMSC-EVs

haMSC-EVs are stable for DP production and long-term storage

Considering that EVs are reportedly unstable at room temperature and sensitive to freeze‒thaw cycles [46], stress tests must be conducted if haMSC-EVs are to be used as drug substances. Thus, we set 3 time points (1 h, 3 h, and 6 h) and 1 or 3 freeze‒thaw cycles to mimic the most extreme scenarios during DP production. The concentration of the particles decreased by less than 20% after 6 h of storage at room temperature or after 3 freeze‒thaw cycles (Fig. 2A), while the proportion of membrane marker-positive vesicles decreased by less than 10% (Fig. 2B). The TNF-α inhibition ratios decreased by less than 15% after 6 h of storage at room temperature and less than 5% after 3 freeze‒thaw cycles (Fig. 2C). The proportions of marker-positive vesicles, and the ratios of TNF-α inhibition of the endpoint samples met the quality criteria (Table 1). These results suggested that the normal DP production process does not affect the characteristics or potency of haMSC-EVs. EVs were reported to be most stable when stored at − 80 °C [47, 48]; however, storage at − 20 °C is more common in clinical practice. To investigate the storage stability at − 20 °C, the drug substances were aliquoted and stored in − 20 °C freezer for 1, 2, 3 or 6 months, with samples stored at − 80 °C serving as controls. The NTA data showed that the particle concentrations decreased by no more than 20% after 6 months of storage at − 80 °C and 3 months of storage at − 20 °C. We found that, the particle concentrations decreased by 50% after the samples were stored at − 20 °C for 6 months (Fig. 2A). However, marker-positive vesicle proportions and TNF-α inhibition ratios were not affected and met the quality criteria after 6 months of storage at − 20 °C (Fig. 2B, C), possibly due to delayed degradation of the markers and effectors. Taken together, the results of the stability study suggest that MSC-EVs are stable for drug development and clinical use.

Fig. 2
figure 2

haMSC-EVs were stable during use and long-term storage. A NTA of haMSC-EVs stored at 25 °C for 1, 3, or 6 h or freeze‒thawed for 1 or 3 cycles to test the in-use stability and stored at -20 °C or -80 °C for 1, 2, 3, or 6 months to test the storage stability. B Membrane marker analysis of haMSC-EVs during use and storage. C TNF-α inhibition ratios of haMSC-EVs during stress tests and storage stability tests. 1 lot was taken for each stress test and 3 lots were tested for storage stability tests

The microRNA profiles of haMSC-EVs were consistent among different lots

Due to their wide regulatory networks, noncoding RNAs, including microRNAs and circular RNAs (circRNAs), have attracted much attention. MicroRNAs have been shown to play important functional roles in MSC-derived EVs [49, 50]. To investigate the microRNA profile of our haMSC-EV product, we constructed small-RNA libraries from 3 lots of haMSC-EVs and performed RNA-seq. We found that haMSC-EVs contain various types of noncoding RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA) and mRNAs (exons and introns). For the microRNA reads, which accounted for 15.91% of the total reads, 0.95% of the reads were mapped to known microRNAs, and 14.96% were identified as novel microRNAs (Fig. 3A). A total of 399 known microRNAs were identified and further compared among the 3 lots of haMSC-EVs, which revealed that 154 (38.6%) microRNAs were shared among the 3 lots of samples, 85 (21.3%) microRNAs were shared between 2 lots, and 160 (40.1%) microRNAs were uniquely identified (Fig. 3B). Next, we used miRanda, TargetScan and RNAhybrid to predict the targets of the common 154 microRNAs. A total of 10,098 target genes were predicted by at least 2 software programs and further analyzed.

Fig. 3
figure 3

haMSC-EV small RNA sequencing and common microRNA target analysis. A Pie chart of small RNA types. B Venn diagram of known microRNAs among 3 lots of haMSC-EVs. C GO analysis of the predicted targets of 154 common microRNAs. D KEGG pathway analysis of the predicted targets of 154 common microRNAs. The dot size represents the number of genes enriched in the corresponding category

Gene Ontology (GO) analysis revealed that up to 71 GO biological process (BP) terms were significantly enriched. The BP terms with the greatest significance were regulation of transcription from the RNA polymerase II promoter, nervous system development, and intracellular signal transduction. A total of 105 GO cellular component (CC) terms were significantly enriched, with the cytosol, nucleoplasm and membrane as the 3 CC terms of greatest significance. There were 40 GO molecular function (MF) terms that were significantly enriched, and the 3 most significant MF terms were protein binding, transcription factor activity and sequence-specific double-stranded DNA binding (Fig. 3C).

KEGG pathway analysis revealed that the predicted target genes were associated with 97 KEGG pathways, including pathways involved in cancer, infection, endocytosis, and cell signaling. The 3 most enriched pathways were pathways associated with cancer, the PI3K-Akt signaling pathway, and pathways associated with human papillomavirus infection (Fig. 3D).

The RNA contents in EVs are relatively low and can be easily lost during isolation. According to previous reports, the common microRNA ratio identified in three repeated samples, isolated with commercial kits or TRIzol, is approximately 50% [51]. Our analysis suggested a slightly lower figure of 38.6% for common microRNAs, showing favorable lot-to-lot consistency. Additionally, these common microRNAs were ranked based on TPM (transcripts per million). The content of the top 20 microRNAs was found to be identical among three lots of haMSC-EVs, with a coefficient of variation (CV) of less than 30% (Table 2). These results indicated that the microRNA contents in haMSC-EVs exhibit suitable lot-to-lot consistency under the current production process. The GO and KEGG results revealed that the microRNAs in haMSC-EVs participate in various cell signaling pathways, biological activities, and infection processes.

Table 2 List of top 20 known microRNAs detected in 3 lots of haMSC-EVs

The haMSC-EVs proteome was stable among different lots

The protein cargo that EVs acquire from their parental cells and carry has a large impact on EV function. The proteomic data of EVs vary depending on the parental cells and isolation methods used [52]. Here, we sought to compare the proteomes of our different haMSC-EV lots to determine the consistency of the isolation process and to further understand their mechanism of action (MOA). Total protein was extracted from 3 lots of haMSC-EVs, digested, TMT-labeled, and subjected to LC‒MS/MS analysis. After peptide mapping, 3819 proteins belonging to 1440 genes were identified. The genes were further compared to genes reported in the available EV database ExoCarta, and 1070 genes (74.3%) were found in this database (Fig. 4A). Our haMSC-EV proteomic data were also compared to 100 “exosomal markers” listed in the ExoCarta database, and 81 proteins were found in this list (Table 3). The TMT method enables relative quantification between samples. We compared the relative content of the proteins between every 2 samples. Proteins with more than 1.5-fold differential expression ratio (P < 0.05) were defined as differentially expressed proteins, and the others were defined as nondifferentially expressed proteins. The results showed a total of 3446 (90.2%) proteins were nondifferentially expressed, 357 (9.3%) proteins were differentially expressed between 2 lots, and only 16 (0.5%) proteins were differentially expressed among all 3 lots of haMSC-EV samples (Fig. 4B).

Fig. 4
figure 4

haMSC-EV protein identification, comparison, and functional analysis of the nondifferentially expressed proteins. A Venn diagram of genes identified in the ExoCarta database and in 3 lots of haMSC-EVs. B Pie chart of differentially and nondifferentially expressed proteins among 3 lots. C GO analysis of the nondifferentially expressed proteins. D KEGG pathway analysis of the nondifferentially expressed proteins. Dot size represents the gene number enriched in the respective category

Table 3 “Exosomal markers” reported in ExoCarta identified in 3 lots of haMSC-EVs

The nondifferentially expressed proteins were subjected to GO and KEGG enrichment analyses. GO analysis indicated that 441 GO BP terms were significantly enriched. The terms with the most significance were cell‒cell adhesion, platelet degranulation and processing, and presentation of exogenous peptide antigen via TAP-dependent MHC class I. A total of 168 GO CC terms were significantly enriched, with the terms of greatest significance being extracellular exosome, cytosol, and focal adhesion. A total of 143 MF GO terms were significantly enriched. The most statistically significant terms were cadherin binding involved in cell‒cell adhesion, protein binding, and GTPase activity (Fig. 4C). KEGG analysis revealed that the proteins were enriched in 67 pathways (P < 0.05), and the most significantly enriched pathways were pathways associated with focal adhesion, pathways involved in cancer, and the PI3K-Akt signaling pathway. There were also pathways related to complement and coagulation cascades, bacterial infection, and chemokine signaling pathways, which were highly enriched (Fig. 4D).

Taken together, the EV attributes of our product are supported by the protein profile, as the results agreed well with those of the ExoCarta database. The relative quantification data demonstrated that the protein cargos of our products were consistent among the lots. In addition, gene function analyses revealed the possible mechanism of the well-reported anti-inflammatory effects of haMSC-EVs[6, 43], which are likely mediated by the proteins involved in bacterial infections and complement cascades.

Four-week repeated toxicity and respiratory toxicity tests in rats to assess the safety of pulmonary haMSC-EV administration

Quality, safety and efficiency are three essential factors of medicine. There are currently no GLP-grade toxicity data for inhaled EVs. Therefore, we conducted a four-week repeated toxicity study of haMSC-EVs in rats by continuous intratracheal administration to evaluate the safety of haMSC-EVs. A total of 118 animals in the 4 groups (98.3% of the total animals tested) survived to the end of the experiment (D62). One animal in the haMSC-EVs-low group died on D26, and one animal in the haMSC-EVs-high group died on D22. Histopathological examination of the two deceased animals revealed minor lung and kidney lesions, which could be caused by damage during administration or by other disorders. In addition, no significant dose-related abnormalities were found in the clinical symptoms, behaviors, eyes tissues, or urine in any of the animals, including the two deceased animals (data not shown). The average body weight and weight gain of the animals in the treatment groups were not significantly different from those of the Control group. At the end of administration period (D29) and the end of the experiment (D62), no significant administration-related toxicity was observed in any of the organ. The results are shown in Fig. 5, Table 4, and Table 5.

Fig. 5
figure 5

haMSC-EVs at three different doses exhibited no influence on the body weight of SD rats

Table 4 Effects of haMSC-EVs on female rats’ organ, hematology and coagulation, serum biochemistry and immune function
Table 5 Effects of haMSC-EVs on male rats’ organ, hematology and coagulation, serum biochemistry and immune function

On the day after the last day of administration (D29), the hemoglobin (HGB) level of male animals in the haMSC-EVs-high group and the albumin (ALB) level of male animals in the haMSC-EVs-low group were slightly lower than those in the Control group (P ≤ 0.01 and P ≤ 0.01 respectively). Otherwise, there were no obvious abnormalities in hematological indices, coagulation, or serum biochemical indices in the treatment groups (Table 4, Table 5).

The detection of lymphocyte markers, which are immune function indicators in the blood did not significantly differ between the treatment and control groups. Gross anatomy (all groups) and histopathological examination (Control group and haMSC-EVs-high group) revealed no abnormal changes, except for one male animal in the haMSC-EVs-medium group, which exhibited multifocal red discoloration of the lungs by the end of administration (D29). Histopathological examination of this animal showed slight bleeding of the lungs and immune cell infiltration, which could be caused by the operation during intratracheal administration.

We further examined BALF from the tested animals. By the end of the administration period (D29), the percentage of neutrophils was significantly higher (P ≤ 0.05), and the percentage of lymphocytes was significantly lower (P ≤ 0.05) in the haMSC-EVs-low group than in the Control group. By the end of the experiment (D62), the percentage of lymphocytes in the haMSC-EVs-high group was significantly lower than that in the control group (P ≤ 0.01). However, the absolute counts of neutrophils and lymphocytes did not significantly change in any animals of the treatment groups compared to those in the Control group (Additional file 3: Table S1, S2). Therefore, we presume that the changes in neutrophil and lymphocyte percentages could be due to normal physiological variation. The remaining indicators did not change significantly. The results are shown in Table 4 and 5.

Animal respiratory function indicators were also examined. The TV in each group was occasionally increased at different time points (P ≤ 0.05 or P ≤ 0.01) after administration, but the difference was not time- or dose- dependent. Other indicators were within the normal range after administration. Moreover, there were no significant differences among the groups at any of the detection time points. The results are shown in Table 6.

Table 6 Effects of haMSC-EVs on respiratory function in rats

In summary, within the range of tested doses, the four-week repeated toxicity and respiratory toxicity studies demonstrated that the pulmonary administration of haMSC-EVs was safe.

Therapeutic effects of haMSC-EVs in an LPS-induced rat model of ALI/ARDS via intratracheal atomization

To verify the results of the in vitro potency assay, we tested the therapeutic effects of haMSC-EVs in an LPS-induced rat model of ALI/ARDS. The animals were randomly divided into four groups: the Control group, Placebo group, haMSC-EVs (6 × 107 particles/rat/time for a total of 2 times) group, and Dexamethasone (1.6 mg/kg/time for a total of 2 times) group. After 24 h, haMSC-EVs reduced the incidence of moderate lung lesions in the model rats compared to that in the Control group (50% vs. 30%) (Fig. 6).

Fig. 6
figure 6

haMSC-EVs alleviated lung injury in an LPS-induced ALI/ARDS rat model. A Histopathology showed that haMSC-EVs decreased the infiltration of inflammatory cells in the alveolar lumen and interstitium and reduced the thickening of the alveolar septum. B haMSC-EVs alleviated the degree of lung lesions (n = 10). − indicates no lesions, ± indicates few lesions, + indicates mild lesions, and + + indicates moderate lesions, +++ indicates severe lesions

To further understand the underlying mechanism, lungs tissues, BALF, and blood were collected for further analysis. At 24 h, compared with those in the Control group, the left lung weight and left lung coefficient in the Placebo group were significantly increased, suggesting pulmonary edema. haMSC-EVs significantly reduced LPS-induced pulmonary edema (0.561 ± 0.026 vs. 0.498 ± 0.033, P ≤ 0.05; 2.329 ± 0.119 vs. 2.144 ± 0.149, P ≤ 0.05) (Fig. 7A). haMSC-EVs also reduced the total protein and total ALB levels in BALF (0.5 ± 0.1 vs. 0.3 ± 0.0, P ≤ 0.01; 0.2 ± 0.0 vs. 0.1 ± 0.0, P ≤ 0.01) (Fig. 7B). The number of inflammatory cells in BALF was also significantly lower in the haMSC-EVs group than in the Control group (Fig. 7C). In addition, haMSC-EVs significantly decreased the serum levels of IL-1β, IL-6 and TNF-α at 4 h (367.5 ± 128.0 vs. 62.1 ± 20.6, P ≤ 0.05; 5222.0 ± 1441.6 vs. 1507.5 ± 153.1, P ≤ 0.05; and 108.4 ± 24.5 vs. 46.0 ± 9.8, P ≤ 0.05) (Fig. 7D). At 24 h, the serum inflammatory factor levels were below the detection limit.

Fig. 7
figure 7

haMSC-EVs alleviated pulmonary edema and inflammation in an LPS-induced ALI/ARDS rat model. A At 24 h, the haMSC-EVs reduced the left lung wet weight and left lung coefficient. B haMSC-EVs reduced the serum ALB and total protein levels in BALF at 24 h. C Inflammatory cell count in BALF at 24 h. D Serum levels of IL-1β, IL-6 and TNF-α decreased 4 h after administration. The serum levels of IL-1β, IL-6 and TNF-α were below the detection limits 24 h after administration. * and ** indicate significant differences compared with the Control group (*, P ≤ 0.05; **, P ≤ 0.01). # and ## indicate significant differences compared with the LPS + Placebo group (#, P ≤ 0.05; ##, P ≤ 0.01)

Based on these results, we concluded that haMSC-EVs alleviated inflammation in LPS-treated rats mainly (or at least in part) by reducing the inflammatory factor levels in BALF and serum.

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