Prognostic implication of downregulated exosomal miRNAs in patients with sepsis: a cross-sectional study with bioinformatics analysis

Abstract Background Despite the understanding of sepsis-induced extracellular vesicles (EVs), such as exosomes, and their role in intercellular communication during sepsis, little is known about EV contents such as microRNA (miRNA), which modulate important cellular processes contributing to sepsis in body fluids. This study aimed to analyze the differential expression of exosomal miRNAs in plasma samples collected from sepsis patients and healthy controls, and to identify potential miRNA regulatory pathways contributing to sepsis pathogenesis. Methods Quantitative real-time PCR-based microarrays were used to profile plasma exosomal miRNA expression levels in 135 patients with sepsis and 11 healthy controls from an ongoing prospective registry of critically ill adult patients admitted to the intensive care unit. The identified exosomal miRNAs were tested in an external validation cohort (35 sepsis patients and 10 healthy controls). And then, functional enrichment analyses of gene ontology, KEGG pathway analysis, and protein–protein interaction network and cluster analyses were performed based on the potential target genes of the grouped miRNAs. Finally, to evaluate the performance of the identified exosomal miRNAs in predicting in-hospital and 90-day mortalities of sepsis patients, receiver operating characteristic curve (ROC) and Kaplan–Meier analyses were performed. Results Compared with healthy controls, plasma exosomes from sepsis patients showed significant changes in 25 miRNAs; eight miRNAs were upregulated and 17 downregulated. Additionally, the levels of hsa-let-7f-5p, miR-331-3p miR-301a-3p, and miR-335-5p were significantly lower in sepsis patients than in healthy controls (p < 0.0001). These four miRNAs were confirmed in an external validation cohort. In addition, the most common pathway for these four miRNAs were PI3K-Akt and mitogen-activated protein kinase (MAPK) signaling pathways based on the KEGG analysis. The area under the ROC of hsa-let-7f-5p, miR-331-3p, miR-301a-3p, and miR-335-5p level for in-hospital mortality was 0.913, 0.931, 0.929, and 0.957, respectively (p < 0.001), as confirmed in an external validation cohort. Also, the Kaplan–Meier analysis showed a significant difference in 90-day mortality between sepsis patients with high and low miR-335-5p, miR-301a-3p, hsa-let-7f-5p, and miR-331-3p levels (p < 0.001, log-rank test). Conclusion Among the differentially-expressed miRNAs detected in microarrays, the top four downregulated exosomal miRNAs (hsa-let-7f-5p, miR-331-3p miR-301a-3p, and miR-335-5p) were identified as independent prognostic factors for in-hospital and 90-day mortalities among sepsis patients. Bioinformatics analysis demonstrated that these four microRNAs might provide a significant contribution to sepsis pathogenesis through PI3K-Akt and MAPK signaling pathway

Successful expansion and cryopreservation of human natural killer cell line NK-92 for clinical manufacturing.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing

Expansion efficacy in the Xuri W25 cell expansion system.

Cells were cultured using a Xuri W25 cell expansion system. The bioreactor was used to control parameters like rock rate, angle, temperature- and gas mix in an automated manner. (A) Expansion efficiency according to cell culture bag capacity. (B) Evaluation of reproducibility of expansion efficiency using NK-92 and TCR-NK-92 cells in the Xuri W25 cell expansion system. The expression level of cell (C) activation receptors, (D) effector molecules, and (E) inhibitory receptors was confirmed after culture up to 12 days. To estimate the change in receptor expression, MFI ratios for each receptor were depicted as measured by flow cytometry. Error bars represent standard error of the mean [25].</p

Cells type used in the study.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.</div

Xuri W25 setpoints.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.</div

Assessment of expansion efficiency of NK-92 cells according to the culture medium.

For NK-92 cells, (A) cell concentration, (B) expansion, (C) viability, and (D) proliferation rate were evaluated every two days. NK cells from cultures of two media were analyzed by flow cytometry for expression of various cell-surface receptors. The expression level of cell (E) activator and (F) effector factors was confirmed after culture up to 12 days. To estimate the change in receptor expression, mean fluorescence intensity (MFI) ratios for each receptor were depicted as measured by flow cytometry. Error bars represent standard error of the mean [25]. p value:* < 0.05, ** < 0.01, **** < 0.0001.</p

Assessment of cell stability after thawing.

Cell stability using three types of cell culture media. (A) Cell expansion efficacy of the NK-92 cell line. (B) Cell morphology as a function of days in culture post-thaw. (C) The efficacy of NK-92 in target cell killing. (D) The expression level of effector molecules in co-culture. Error bars represent standard error of the mean [25]. p value: *** < 0.001, **** < 0.0001.</p

Assessment cell viability after cryopreservation for NK-92.

Cell viability was evaluated according to the freezing rate. Cell viability after thawing by type of cryoprotective agents (A) frozen at a rate of -0.5°C/min, (B) a rate of -1.0°C/min, and (C) a rate of -2.0°C/min. Cell viability was evaluated according to nucleation induction during cell freezing and viability results according to cell concentration. (D) Cell viability after thawing as a function of cell concentration, (E) Cell viability after thawing according to cell concentration induced nucleation. Cell viability was assessed after long-term storage of frozen cells. Cell viability and expansion efficacy of (F) 1 month, (G) 3 months, and (H) 6 months after storage of frozen cells in liquid nitrogen vapor phase. Evaluation of preservation efficacy in cryo-bags (I)Assessment cell viability after cryopreservation for NK-92. Cell viability was assessed after long-term storage of frozen cells. Cell viability and expansion efficacy of 1 month, 3 months, and 6 months after storage frozen cells. p value: ns > 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.</p

Evaluation of cell aggregation according to culture media after cell thawing.

NK-92 cells were frozen using seven types of cryoprotective agents. After thawing the frozen cells, (A) Cultured in Xuri T-cell media after 24 hours. (B) Cultured in X-vivo 10 media to observe cell aggregation after 24 hours. (TIF)</p

Assessment of cytokine production efficacy of NK-92 cells according to the culture medium.

(A) NK-92 cells cultured in three distinct media were subjected to flow cytometry analysis to evaluate the expression of effector molecules. To quantify the alterations in expression levels, we represented the MFI ratios for each individual molecule as determined through flow cytometry measurements. (TIF)</p