Table 4. Summary of in-vitro studies assessing the exosome effect on human corneal cell line, salivary glands and mononuclear cells.

Study	Experimental target	Source of exosomes	Exosome volume	Exosome size	Exosome delivery method	Exosome content/ markers	Outcomes measured	Results
Yu et al. 2020	Human corneal epithelial cells exposed to hyperosmolar medium for 24 h	Human adipose tissue MSCs	1μg/μL	100 nm	Co-incubated with HCECs for 4h	TSG101, CD63, ALIX	TUNEL assay, cell viability assay, NLRP3, ASC, capase− 1 and IL− 1b levels	TUNEL positive cells increased in hyperosmotic medium (17.42 +/- 4.20% vs 1.55 +/−0.55 %), that decreased after treatment with exosomes, with TUNEL positive cells reducing to 4.88 + /−2.49 %. Increased mRNA expression of NLRP3, ASC, capase−1 and IL−1b post hyperosmotic stress reduced by exosome treatment
Rui et al.2021	Mouse CD4 + T cells	Olfactory ectodermal mesenchymal stem cells (OE-MSC), Bone marrow mesenchymal stem cells (BM-MSCs) derived from the tibia and femur		50−150 nm	OE-MSC-Exos or BM-MSC-Exos were co-incubated with MDSCs for 2 days, and then co-cultured with mouse CD4 + T cells.	CD9, CD63	CFSE fluorescence to evaluate CD4 + T cells by flow cytometry, western blot analysis	OE-MSC-Exos promoted expansion of MDSCs much more than BM-MSC-Exos. OE-MSC-Exos led to enhanced suppressive effect of MDSCs on CD4 + T cell proliferation.
Kim et al. 2021	TLR−4 stimulated splenocytes from adult male BALB/c mice	Human induced pluripotent stem cells from MSCs, exosomes from P5 and P15 iMSCs	1.5×10^10 particles in 100 μL PBS	50−300 nm	Co-incubation	CD9, CD63, CD81	Cytokines secreted by activated T lymphocytes, mRNA expression of markers for IL−17, IL−6, IL-1, TGF-b1, IFN-gamma. Protein profiling of P5 and P15 iMSC EVs	P5 iMSC EVs were more effective than P15 iMSC EVs in suppressing the secretion of pro-inflammatory Th1 and Th17 cytokines, IFN-gamma, IL− 6, IL−17 and increasing TGF-b1.
Li et al. 2022	Differentiated human macrophages	Human umbilical cord MSCs	na	50−150 nm	Differentiated macrophages were co-incubated with exosomes 5 μg/mL stained with PKH26 for 48hrs.	CD81, CD9, CD63, TSG101	mRNA and protein markers	mRNA levels of M2 markers, protein markers of M2 macrophages and expression of Treg markers were all increased in the exosomes group compared to control. Exosome educated macrophages when cultured with CD4 + T cells led to significant repression of the CD4 + T cells.
Xing et al. 2022	Human peripheral blood mononuclear cells (PBMCs)	Human labial gland mesenchymal stem cells (LGMSCs)	30μg/mL	Median 121 nm, range 80−497 nm	Co-culture of PBMCs and LGMSCs at a ratio of 10:1, in a subset an exosome inhibitor GW4869 was also added.	CD9, CD63, Psg101	Flow cytometry for B cell markers CD19, CD20, IgD, CD38, CD27, CD24. RNA isolation and qRT-PCR	PBMCs with LGMSC-Exos led to reduced CD19, CD20, CD24, CD38 plasma cells, this effect was less pronounced with the exosome inhibitor GW4869
Guo et al. 2022	Human corneal epithelial cells cultured in hyperosmotic culture medium to simulate DED	Human umbilical cord MSCs	na	80−180 nm	Co-culture of MSC-EVs with HCECs	CD9, CD63 and CD81 (Calnexin was negatively expressed)	Flow cytometry to detect Th17 cells, RT-PCR to detect mRNA expression of inflammatory cytokines, immunofluorescence to detect dendritic cells	MSC-EVs were protective to HCECs under hyperosmotic stress by suppressing inflammatory cytokines TNF-alpha, IL−6, IL−1b. MSC-EVs at concentrations ranging between 10 and 50μg/mL were protective for HCECs
Tian et al. 2023	Human corneal epithelial cells	Murine bone marrow, Ce (NO3)3·6H2O was incorporated into the MSC-Exos to form MSCExo-Ce	20−40μ/mL	100−150 nm	Co-incubation	CD 48, CD 63, TSG101	ROS scavenging activity assay e, uptake of MSC-Exo on HCECs via Cy5 staining, in vitro scratch wound assay, mitochondrial ROS scavenging, cytotoxicity study of MSC-Exo-Ce	MSC-Exo-Ce demonstrated superior ROS scavenging ability compared to MSC-Exos. HCEC uptake of exosomes was not impaired by Ce coupling. HCEC re-epithelialisation was significantly improved with MSC-Exo-Ce, with a smaller wound surface area (27.63) compared to MSC-Exo (34.83) or PBS (77.53).
Li et al. 2021	Peripheral blood mononuclear cells from SS patients	Human Labial gland mesenchymal stem cell exosomes (LGMSC-Exos)	30μ/mL		LGMSCs and LGMSC-Exos co-cultured with PBMSCs for 72hrs (1:10)	CD 9, CD 63, CD 81	Percentage of Th17 and Treg, cytokines in the supernatant	LGMSC and LGMSC-Exos significantly suppressed the cytokines IL−17A, IFN-γ, IL−6, and TNF-α and increased TGF-β and IL-10.
Chu et al. 2023	Damaged labial gland tissue from SS patients	Human exfoliated deciduous teeth	na	137 + / −6.2nm	Co-cultured for 24 hrs	CD9, CD63 and HSP70	Cell death markers ACSL4, KEAP1, RIPK3	Cell death markers expression downregulated in SS labial glands. Exosomal miRNA associated with cell death pathways were identified to be KRAS, HRAS, MEK1/2 and the downstream signalling molecule p-ERK1/2
Du et al. 2023	Human submandibular gland tissue	Human exfoliated deciduous teeth	200μ/mL	126.5 + / −5.7	Co-cultured for 24hrs	CD9, CD63, CD81, HSP70	Exosome uptake into the glandular cells	PKH− 26-stained exosomes were found in the cytoplasm of SMGC6 cells, acinar cells and duct cells.
Hu et al. 2023	IFN-γ treated salivary gland epithelial cells	Human dental pulp stem cell (DPSC)	5, 20 and 80μg/mL	30−150 nm	Different concentrations of DPSC-Exos were added to SGECs for 24, 48 and 72 h	CD63, ALIX, TSG101	AQP5, GPER expression	Increased proliferation, AQP5 and GPER expression in the IFN-γ treated SGECs with 20 and 80μg/mL DPSC-Exos.
Zhou et al. 2023	CD19 + B cells were cultured under Germinal Centre B polarising conditions	Myeloid derived suppressor cells from the spleen of tumour bearing C57BL/6 mice		50−150 nm	Co-cultured for 96 h with CD19 + B cells	CD63, CD19, TSG101, but not calnexin	Differentiation of B cells	Exosome treatment suppressed differentiation of B cells, decreased mRNA and protein levels of Bcl−6. miR− 10A− 5p is an exosome miRNA thought to be involved in regulating Bcl−6 expression
Zhao et al. 2023	Isolated splenocytes from 4-month-old female NOD.B10. H2 mice, model of primary SS	Human induced pluripotent stem cells from MSCs, early passage PD15 and late passage PD45	3×10’9 particles/mL		Early and late passage EVs were labelled with PKH26 and co-incubated with splenocytes for 3 hrs		Flow cytometry analysis for PKH26, macrophage, T cell and B cell markers	Macrophages were the major population > 70 % uptaking iEVs both early and late passage, Young EVs (early passage PD15) promoted M2 polarisation of splenic macrophages that was maintained 2 weeks after the last IV treatment in PD15 (young) but not with PD45 (aged) EVs
Ma et al. 2023	Human corneal epithelial cells monolayer with scratch assay. Incubated with AA, mExo, mExo@AA and saline for 12 h	Mouse mesenchymal stem cell derived exosomes with and without ascorbic acid coupling	0.01−1.0 mg/mL	100−150 nm	Unknown	ALIX, beta-actin, TSG 101 positive, negative for GM130	Re-epithelialisation of the monolayer of HCECs, ROS scavenging ability, macrophage immunophenotype	mExo@AA accelerated wound closure compared to saline, mExos and AA alone, increased ROS scavenging ability. mExo@AA showed the greatest increase in M2 phenotype (anti-inflammatory) of macrophages; mExo and mExo@AA reduced pro-inflammatory cytokines IL−6 and IL-1β, with mExo@AA and mExo having similar effects.
Zhou et al. 2023	CD19 + B cells were cultured under Germinal Centre B polarising conditions	Myeloid derived suppressor cells from the spleen of tumour bearing C57BL/6 mice		50−150 nm	Co-cultured for 96 h with CD19 + B cells	CD63, CD19, TSG101, but not calnexin	Differentiation of B cells	Exosome treatment suppressed differentiation of B cells, decreased mRNA and protein levels of Bcl−6. miR− 10A− 5p is an exosome miRNA thought to be involved in regulating Bcl−6 expression

MSC-Mesenchymal Stem Cell, PBS − Phosphate Buffer Solution, SS − Sjögren’s Syndrome, DED-Dry Eye Disease, HCEC − Human Corneal Epithelial Cell, TUNEL - terminal deoxynucleotidyl transferase dUTP nick end labeling, ASC - Apoptosis-associated speck-like protein containing a caspase recruitment domain, CFSE - Car-boxyfluorescein succinimidyl ester, MDSC − Myeloid Derived Stem Cells, qRT-PCR - Qualitative Reverse Transcription Polymerase Chain Reaction, ROS − Reactive Oxygen Species, SGEC − Salivary Gland Epithelial Cell, AQP5 − Aquaporin 5, GPER − G-protein coupled estrogen receptor, NOD − Non-Obsese Diabetic, AA − Ascorbic Acid