ABSTRACT Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical

applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of

Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use _in vitro_ or _in vivo_, thus

promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in

contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. SIMILAR CONTENT BEING VIEWED BY OTHERS BIOMOLECULAR PHENOTYPING AND HETEROGENEITY

ASSESSMENT OF MESENCHYMAL STROMAL CELLS USING LABEL-FREE RAMAN SPECTROSCOPY Article Open access 23 February 2021 ISOLATION AND CHARACTERIZATION OF EXTRACELLULAR VESICLE SUBPOPULATIONS FROM

TISSUES Article 25 January 2021 MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES FOR IMMUNOMODULATION AND REGENERATION: A NEXT GENERATION THERAPEUTIC TOOL? Article Open access 04 July

2022 INTRODUCTION Extracellular vesicles (EVs) are a heterogeneous group of membrane-bound vesicles that are constitutively released by cells of different tissue origins. Past controversies

concerning nomenclature have now been resolved by the scientific community, which defines EVs as the group of particles made up of exosomes, microvesicles and apoptotic bodies1. Exosomes

(30–100 nm) and microvesicles (up to 1000 nm) differ in size and cellular origin, but both mediate intercellular communication within a tissue and among organs thanks to body fluid

transportation1. As is the case for most body cells, part of the secretome of mesenchymal stromal cells (MSCs) includes exosomes and microvesicles, which are currently being investigated

because of their striking regenerative and immunomodulating potential. The bioactive molecules loaded onto/into EVs are involved in the paracrine effects of stem cells, and even the membrane

constituents of vesicles seem to trigger intracellular protective/regenerative pathways in recipient cells2. It has been suggested that MSC-derived EVs may be sometimes even more

therapeutically valuable than whole cells, because of their remarkable handling advantages, which can accelerate their clinical application in the so-called _cell therapy without cells_ 3.

The possibility of overcoming the cell therapy drawbacks of having to administer living, replicating and difficult to control cells is currently one of the main challenges facing

regenerative medicine, and EVs can be an effective means of stimulating the restoration of organ function through tissue regeneration and repair in the context of an integrated strategy of

_regenerative rehabilitation_ 4. Over the last ten years, many studies have demonstrated the role that MSC-derived EVs can play in tissue repair and immunomodulation5, 6 and, in 2014, EVs

ability to influence the activity of recipient cells and regulate immune responses was successfully exploited in a patient undergoing allogeneic hematopoietic stem cell transplantation who

developed therapy-refractory graft-versus-host disease7. Their regenerative potential has also been assessed in _in vitro_ and _in vivo_ models of many diseases affecting heart8,9,10,

kidney11,12,13, liver14, 15, bone and cartilage16, 17, muscle18, skin19, and central nervous system20,21,22,23. However, there are still concerns about the effect that the source of MSCs and

cell culture conditions can have on EV production and characteristics as there is no standardised and optimised method for isolating and characterising EVs. Furthermore, the technical

restraints of current techniques have limited their potential use in regenerative medicine24, 25 by preventing reproducible quality and safety assessments26. The aim of this study was to

test Raman spectroscopy (RS) as a label-free, non-destructive, sensitive, rapid and automatable means of carrying out the bulk characterization of EVs. This technique provides a spectrum

that qualitatively and quantitatively describes the chemical composition of a sample and thus avoids the need for specific protein biomarkers. It has been widely used in the pharmaceutical

industry as a mean of verifying raw materials and quality controlling drug production, and we suggest it could help in purity and quality checking vesicle suspensions. It has already proved

its value by characterising a wide range of cells and tissue samples for the purposes of basic research, and as an innovative alternative to classic, time-consuming and operator-dependent

diagnostic methods27,28,29,30,31,32,33,34. In the field of regenerative medicine, it has been used to analyse undifferentiated and differentiated human and murine embryonic stem

cells35,36,37 and to monitor MSCs stimulated towards osteogenic differentiation38, 39. Efforts have also been made to develop Raman-based methods for the individual characterisation of human

vesicles40, 41, but although these have provided information at single-vesicle level, they are still far from being used diagnostically. What is required to allow the immediate

transferability of EV research to clinical practice is a procedure that allows i) the rapid characterisation of a sample before its use _in vitro_ or _in vivo_; ii) the identification of

fingerprints of the EV populations used for regenerative purposes in order to determine the best experimental settings and compare results from different cell sources; and iii) the routine

application of the analysis. The third point should be favoured by the current availability of portable Raman spectrometers that can automatically scan and analyse complex samples, which

could bring Raman analysis easily in the reach of most laboratories. RS is much more suitable for achieving these goals than the widely used techniques of immunoblotting, cytofluorimetry and

spectrometry because it can provide reproducible results quickly and in a label-free manner, and only requires tiny sample volumes in comparison with the large amounts needed by other

methods, which cannot easily cope with the nanoscalar dimensions of exosomes. This study provides the first Raman-based characterisation of the EVs of human MSCs isolated from bone-marrow

(bone marrow mesenchymal stromal cells, BM-MSCs) and subcutaneous adipose tissue (adipose tissue mesenchymal stromal cells, ASCs). The results were compared with those obtained using EVs

released by dermal fibroblasts (DFs), in order to verify the ability of Raman analysis to distinguish vesicles from undifferentiated and differentiated cells, and gain insights into the

biochemical features of EVs from different sources. Multivariate analysis was used to assess spectral differences and automatically distinguish the three groups. In addition, given the

growing body of evidence concerning the pivotal role of lipids in mediating EV functions42, we also evaluated the contribution of lipid membrane constituents to the Raman spectra. Our

findings provide evidence supporting the use of RS for the routine characterisation of MSC-derived EVs before their _in vitro/in vivo_ application. RESULTS EV CHARACTERISATION EVs were

isolated from BM-MSCs, ASCs and DFs following a multi-step ultracentrifugation protocol43 and characterised by immunoblotting and transmission electron microscopy (TEM) to verify their

peculiar features as suggested by the International Society for Extracellular Vesicles (ISEV)44. Immunoblotting confirmed the presence of EVs carrying flotillin-1, CD63 and CD9, and a

significant reduction in calnexin-positive vesicles (Fig. 1A). The TEM images (Fig. 1B–D) confirmed the typical morphology of the EVs, whose ultrastructure and size were consistent with

published data45. The vesicles in all of the samples were round (Fig. 1B–D) and their general mean diameter as calculated on the TEM images was 46.5 nm (±15.8 nm) with slight differences

among cell groups. Supplementary Figure S1 shows a box plot with all of the recorded measurements. RAMAN SPECTROSCOPY BIOCHEMICAL OVERVIEW OF EVS Freshly isolated EVs were analySed by RS in

the spectral ranges of 500–1800 cm−1 and 2600–3200 cm−1, the most significant regions of the Raman spectrum for biological specimens. The spectra were obtained from random spots of air-dried

drops of EV suspension and, given the size of the laser beam, we speculate that every spectrum described the biochemical features of small clusters of aggregated EVs. Figure 2 shows

representative mean Raman spectra (±1 standard deviation) of the vesicles isolated from the supernatants of BM-MSCs, ASCs and DFs. Each mean spectrum represents the average of 40–50

independent recordings obtained from all of the donors of the same cell type. The overall homogeneity in the spectra from the same tissue source underlines the reproducibility of the

analytical method, which is not affected by the intrinsic inter-individual variability of donors. The spectra showed characteristic Raman bands of nucleic acids (NAs, 720–820 cm−1),

phenylalanine (Phe, 1003 cm−1), lipid and protein markers such as CH and CH2 groups (bands respectively centred at 1450 cm−1 and 2940 cm−1) (Fig. 2 and Table 1). In particular, lipids made a

large contribution, which is in line with previously reported spectroscopic evidence40, 41, 46. Lipid content was characterised by peaks attributable to cholesterol and cholesterol ester

(537; 702; 1130; 1442 cm−1) and peaks of varying intensity corresponding to the C-C stretch (around 1100 cm−1) and CH, CH2, and CH3 bonds (in the spectral range 2600–3200 cm−1). In addition,

the areas usually assigned to NA bases (718; 748; 782 cm−1) and phosphate backbone (785; ~1060 cm−1) were variably prominent in the three average spectra. This is in line with many data

demonstrating that EVs contain intact mRNA, long non-coding RNA, miRNA and other forms of RNA loaded into EVs42. The recurrent peaks attributable to proline/hydroxyproline (853; 920; 1206 

cm−1) and tryptophan (752–760; 1208; 1360; 1555 cm−1) may be related to differences in cell metabolism and responses to serum-deprived culture conditions. Proline is known to be a signalling

molecule and a sensor of cellular energy status when responding to metabolic stress47, and the kynurenine pathway of tryptophan has been reported as being involved in the immunosuppressive

effects of MSCs48. Comparison of the average spectra revealed many differences between the cytotypes (highlighted in bold characters in Table 1), suggesting discrepancies in the panel of

protein biomarkers and lipid content of vesicles, although it is difficult to attribute divergences in peak intensity to specific molecules. The presence of a 1127 cm−1 peak seemed to

distinguish ASC spectra from those of both BM-MSC and DF EVs. The comparison of ASC and DF data highlighted minor divergences in the spectral range 2600–3200 cm−1, which is greatly

influenced by lipid molecules thus indicating similarities in the lipid content of ASC- and DF-derived vesicles. LIPID MEMBRANE CONSTITUENTS ACCOUNT FOR SPECTRAL DIFFERENCES Principal

Component Analysis (PCA) was used to simplify the original data (n = 198) and all of the spectra were collectively represented by their principal components (PCs). Starting from PC1 (which

accounted for 37.1% of total variance), the subsequent PCs describe differences in the Raman fingerprint that were progressively less prominent (Supplementary Table S1 and Supplementary Fig.

 S2). The first 2 PCs (Fig. 3A) were used to build the scatter plot shown in Fig. 3B. Combined analysis of the scatter plot and the PC1 and PC2 spectra revealed that the positive loadings in

the PC1 spectrum mainly describe the biochemical features of ASC-derived EVs, positive peaks in PC2 represent BM-MSC vesicles rather than ASC or DF vesicles. One-way ANOVA performed on PC1

and PC2 scores demonstrated that the means of each group were significantly different (Prob > F < 0.05), despite within-group variance (Supplementary Table S2). Based on the simple

premise that a spectrum from a mixture of chemical ingredients is a mixture of the spectra from the pure ingredients, the PC1 and PC2 loadings were least squares fitted (classical least

square (CLS) fitting) with specific reference spectra to investigate one possible cause of the observed spectral differences, following a previously reported procedure40. As we observed that

the most variable spectral intervals in PC1 and PC2 were related to lipids, the membrane components cholesterol (Chol), ceramide (Cer), sphingomyelin (SM), phosphatidylcholine (PCh),

phosphatidylethanolamine (PE), phosphatidic acid (PA), and monosialotetrahexosylganglioside (GM1, reference molecule for monosialoganglioside family) were used for CLS fitting. Lipid

reference molecules were preferred to protein markers because proteins spectra are dominated by backbone conformation signals, whereas lipids have more specific and defined peaks and can be

more easily distinguished by RS. The resulting CLS fitting scores reported in Table 2 described the relative contribution of each standard molecule to PC1 and PC2 loadings, thus their

contribution to the observed spectral differences between the three cytotypes. Figure 3C depicts the fitting coefficients in a bar graph, making apparent that SM and ganglioside (GM1)

contribute to the shape of PC1 and PC2 loadings more than the other reference molecules. Similarly, PCh is the phospholipid which contributed most to fit the shape of PC1. In particular, the

positive score attributed to SM after PC1 fitting demonstrated that it made a contribution to the spectrum of ASC-derived EVs, and this was further underlined by the negative SM score after

PC2 fitting. On the contrary, GM1 and PCh were assigned a negative CLS fitting score in the case of PC1, suggesting their presence within the membrane of EVs from BM-MSCs and DFs rather

than ASC-derived vesicles. After CLS fitting, the scores assigned to Chol, Cer, PE, and PA suggest their presence in the EVs from all three source cells, although they do not greatly

contribute to the differences between EVs. It has to be noted that the considered lipids are only few of the constituents of vesicles, for this reason our results should be considered as

hints for future studies aimed at verifying the exact membrane composition of vesicles. RAMAN SPECTROSCOPY CAN DISTINGUISH BM-MSC, ASC AND DF EVS WITH 93.7% ACCURACY The first 25 PCs were

used for Linear Discriminant Analysis (LDA), which made possible to verify the ability of the method to identify between-group differences by maximising the variance among classes while

minimising intra-class variability. The results showed that RS clearly distinguished the biochemical fingerprints of the three groups. After leave-one-out cross-validation, the PCA-LDA model

showed that the overall accuracy of the model was 93.7% and that its accuracy in distinguishing DFs from MSCs was 92% (Table 3). The LDA scatter plot (Fig. 4) revealed that the spectra of

the ASC-derived EVs fell into a region that was clearly separated from those of the BM-MSC EVs. Although there was a limited overlap between the DF and ASC derived vesicles, RS distinguished

their sources with a high degree of statistical confidence (Wilks’ Lambda Test, p < 0.001). Details concerning the distribution of the individual donor spectra are shown in Supplementary

Figure S3. DISCUSSION The possibility of using regenerative medicine to treat diseased, damaged or aged tissues and restore organ function without side effects is one of the main challenges

facing modern medical science, and so is no surprise that the discovery of the regenerative potential of EVs released by MSCs has aroused great interest. However, the main obstacle to the

clinical use of vesicles is the lack of a robust and standardised method of characterising them5. In this study, we investigated the biochemical fingerprints of MSC-derived vesicles

originating from different tissues and compared them with those of terminally differentiated dermal fibroblasts. Our findings demonstrate the ability of RS to identify tissue-related

fingerprints for vesicles released by MSCs and fibroblasts without the use of any label. This is the first time that Raman analysis has been used to provide a biochemical overview of

MSC-derived EVs from a limited volume of EV suspensions. As previously reported in relation to other types of vesicles40, 41, 49, our data on MSC-derived EVs confirm the ability of RS to

reveal the presence of the main EV constituents in a single repeatable spectrum. Although we cannot exclude the possible presence of a limited amount of soluble factors other than vesicles

in our suspension, the reproducibility of the results and the main peak attributions suggest the purity of the samples. The main finding of this study is that RS can clearly distinguish not

only vesicles from MSCs and terminally differentiated fibroblasts, but also vesicles of MSCs from bone marrow and adipose tissue. Although there are protein markers that define a stem cell

phenotype exist, a straightforward distinction between bone-marrow and adipose-tissue MSCs, based on biological and functional features, is still difficult to be obtained50. The ability of

RS to highlight unique, tissue-specific features of vesicles should therefore assist scientists working with stem cells. Even if assessing the possible correlation between biochemistry and

function goes beyond the scope of this study, the biochemical variations observed provide suggestions for further investigations into the functional differences of EVs from multiple MSC

types and sources for which there is still not a definite marker51, 52. Analysis of the spectra of MSC- and DF-derived EVs revealed that lipids made a substantial contribution to the Raman

signals, as previously reported40, 41, 49. The prominence of membrane constituents in determining the fingerprints of vesicles is in line with the growing body of evidence demonstrating that

lipids play a crucial role in the formation of EVs1 and the fulfilment of their signalling functions42. It is known that a number of specific lipids are typically associated with lipid

rafts and enriched in vesicles that inherit the plasma membrane composition of their cell of origin. In particular, cholesterol and sphingolipids are preferentially included in EV membranes

and may be involved in the formation of vesicles and in their stability in the extracellular environment42. There is also evidence that lipids are involved in BM-MSC responses to a strongly

pro-inflammatory environment53. Furthermore, it is possible that direct membrane interactions between vesicles and recipient cells is one of the mechanisms of action of EVs, as has already

been demonstrated in the case of whole cells2. On the basis of CLS fitting results, we hypothesised that gangliosides, phosphatidylcholine and sphingomyelin directly contributed to the main

spectral differences between the considered EVs. Our data are in agreement with those of a recent proteomic and lipidomic study demonstrating how sphingomyelins, ceramides, cholesterol and

phosphatidylcoline were enriched in the exosomes of BM-MSCs in comparison with other cell types54, but there is still a lack of data concerning the membrane composition of ASC-derived

exosomes. Our observation that GM1 also contributes to the recorded spectra of BM-MSC vesicles is in line with the reported functional role of gangliosides in regulating the proliferation

and neuronal differentiation of MSCs55, 56. Similarly, it is known that ceramides and ceramide-containing lipids are involved in many of the pathways mediating immune responses, and that

they modulate the adipogenic differentiation of MSCs57. Despite the reported functional significance of PA in the biogenesis and release of exosomes, our data did not reveal any significant

difference of PA content in the EVs derived from the three cell types, as has also been noticed by Haraszti _et al_.54 Further lipidomic studies are needed to verify the exact membrane

composition of MSC-derived vesicles and to establish the role of lipid species in mediating vesicle function. It is important to mention one limitation of our study related to the sex

mismatch of our MSC donors. It is known that MSC activity and recipient responses are influenced by the sex of both donor and recipient because of circulating hormones58, but, to the best of

our knowledge, no specific study has been published concerning sex-related variations in the function of EVs derived from cultured cells. Studies evaluating the efficacy of MSC-derived EVs

_in vitro_ and _in vivo_ rarely refer to the sex of the donors, but this is very useful and should be always clearly indicated together with donor age59. Future in-depth analyses of larger

donor cohorts should evaluate age and sex-related differences in EV function and chemical composition. In conclusion, our findings demonstrate that RS can determine the chemical content of

EVs in a label- and sample processing- free manner. The proposed method can be immediately transferred into laboratory practice as it allows the bulk characterisation of vesicle suspensions

before their use _in vitro_ or _in vivo_. As independent MSC-derived EV preparations can have different therapeutic potentials, the overall characterisation of vesicles offered by Raman

spectroscopy might become a pivotal quality check for comparing data coming from different experiments or research labs, and thus hasten the clinical application of stem cell-derived

products. METHODS All of the relevant experimental data have been submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV170012)60. CELL CULTURES Human BM-MSCs were isolated from the

residual bone marrow cells of healthy bone marrow (BM) transplantation donors (3 male donors, age range: 17–20 y/o) after approval by the Institutional Review Board of San Raffaele Hospital.

Human ASCs and DFs were isolated from waste materials of abdominoplasty and liposuction procedures performed at IRCCS Galeazzi Orthopaedic Institute (subcutaneous adipose tissue of 4 female

healthy donors - age range: 35–58 y/o - and de-epidermised dermis of 3 female healthy donors – age range: 26–46 y/o-, respectively). Tissues were collected following the procedure PQ

7.5.125 regarding waste materials to be used for research purposes, version 4 dated 22.01.2015, approved by the same institute. Written informed consent was obtained from all of the patients

in accordance with the ethical principles of the Declaration of Helsinki. All of the samples were anonymised and no information or images that could lead to identification of a study

participant might occur. All experiments were performed in accordance with the relevant guidelines and regulations of San Raffaele Hospital and IRCCS Galeazzi Orthopaedic Institute. Cells

were isolated following previously described protocols61,62,63. Briefly, mononuclear cells from BM aspirates were isolated by means of density gradient centrifugation (Ficoll 1.077 g/ml;

Lympholyte, Cedarlane Laboratories Ltd., Burlington, Canada) and plated in non-coated 75–150 cm2 tissue culture flasks (BD Falcon, Franklin Lakes, NJ, USA) at a density of 160,000/cm2 in

complete culture medium: DMEM (Euroclone, Milan, Italy) supplemented with 10% ultracentrifuged foetal bovine serum (Gibco, Life Technologies LTD, Paisley, UK), penicillin 50 U/ml, 50 µg/ml

streptomycin and 2 mM L-glutamine (L-Glu, Euroclone). Cultures were maintained at 37 °C in a humidified atmosphere, containing 5% CO2. After 48-hour culture, non-adherent cells were removed.

The ASCs were isolated from adipose tissue samples following digestion with 0.75 mg/ml type I Collagenase (250 U/mg, Worthington Biochemical Corporation, Lakewood, NJ, USA) and the

filtering of the stromal vascular fraction. The DFs were obtained from de-epidermised dermis fragmented and digested with 0.1% collagenase type I. The ASCs and DFs (plating density: 105

cells/cm2) were cultured (37 °C, 5% CO2) in complete culture medium. The medium was replaced every other day and, at 70–80% confluence, the cells were detached with 0.5% trypsin/0.2% EDTA,

plated (BM-MSC plating density 4,000 cells/cm2; ASC plating density 10,000 cells/cm2; DF plating density 5,000 cells/cm2) and expanded. Once at 80–90% confluence, cells at 3rd–4th passage

were washed twice with DMEM, kept for one hour in serum-free DMEM (phenol-free DMEM supplemented with 2 mM L-glutamine, 50U/ml penicillin, 50 µg/ml streptomycin) and then cultured for 72 

hours in serum-free DMEM. EXTRACELLULAR VESICLE ISOLATION In order to avoid the presence of RS-visible isolation reagent residues in the EV suspension, the vesicles were isolated from

cell-conditioned medium (CM) by means of differential centrifugation, as previously described43. Briefly, after 72 hours of starvation, the medium conditioned from approximately 6 × 106

cells was centrifuged at 800 g for 10 min to remove non-adherent cells and then at 2,500 g for 15 min to remove potential apoptotic bodies. CM was then ultracentrifuged for 70 min at 100,000

 g (L7–65; Rotor 55.2 Ti; Beckman Coulter, Brea, CA, USA) at 4 °C, and the pellet was re-suspended in sterile saline solution and ultracentrifuged again. The collected EV suspension

(approximately 500 µl) was kept at 4 °C before making Raman and TEM analyses, and then frozen. WESTERN BLOTTING Immunoblotting was performed to characterise the EVs as suggested by ISEV

minimal experimental requirements44. The EV pellets were re-suspended in SDS sample buffer with protease inhibitors64. Electrophoresis was performed under reducing conditions, and then

proteins were transferred to nitrocellulose membrane. The antigens were probed with anti-flotillin-1 (BD Transduction Laboratories™, San Jose, CA, USA), anti-CD63 and anti-CD9 (System

Biosciences, Palo Alto, CA, USA), and anti-calnexin (endoplasmic reticulum protein used as negative control, clone C5C9, Cell Signaling Technology, Danvers, MA, USA). As secondary antibodies

goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-rabbit (System Biosciences) conjugated with HRP were used. Cell lysates were considered as the control for the

specificity and working conditions of the considered antibodies. TRANSMISSION ELECTRON MICROSCOPY AND SIZE MEASUREMENT For the TEM visualisation of EVs, 5 µl of purified exosomes were

absorbed on Formvar carbon-coated grids for 10 min. The drops were then blotted with filter paper and negatively stained with 2% uranyl acetate (5 μl) in aqueous suspension for 10 min.

Excess of uranyl was removed by touching the grid to a filter paper. The grids were dried at room temperature and examined with a transmission electron microscope (Leo 812AB, Zeiss,

Oberkochen, Germany) at 80 kV. The TEM images obtained in order to verify EV ultrastructure were used to assess vesicles’ size using the particle analysis tool of ImageJ software (National

Institutes of Health, Bethesda, MD, USA). At least 30 measurements per sample were done. RAMAN SPECTROSCOPY Freshly isolated EVs were analysed by means of Raman microspectroscopy (LabRAM

Aramis, Horiba Jobin Yvon S.A.S, Lille, France) equipped with a diode-pumped solid-state laser operating at 532 nm and a Peltier-cooled CCD detector. 5–10 µl drops of EV suspension were

deposited on a calcium fluoride slide and allowed to air dry. All of the measurementS were performed with 50× objective (NA 0.75, Olympus, Tokyo, Japan), 1800 grooves/mm diffraction grating,

400 µm entrance slit, and confocal mode (600 µm pinhole) in the spectral ranges 500–1800 cm−1 and 2600–3200 cm−1. Accumulation times were 2 × 10 s per spectrum. The Raman shift was

calibrated automatically using LabSpec 6 software (Horiba) using zero order line and Si line of a Si reference sample. In order to capture the spectra randomly, maps of about 150 µm2 (with

lateral steps of 20–30 µm) were acquired in the centre and at the borders of the air-dried drops. Before analysing the data, a two-class hierarchical clustering analysis (HCA) of the Raman

maps was made in order to distinguish the spectra relating to vesicles from those related to background. At least 10 independent replicates of the Raman spectra were obtained for every donor

of the different cell types. DATA ANALYSIS Baseline correction was made using LabSpec 6 processing tool by fitting all spectra with a sixth order polynomial curve in order to remove

autofluorescence and background before unit vector normalisation. Post-acquisition calibration was carried out on normalised spectra, in order to compensate for possible thermal drifts.

Principal component analysis (PCA) of the normalised and aligned spectra was made in order to reduce the dimension of the data and describe their major trends. The provided principal

components (PCs) represent differences in the spectra of vesicles from the three cytotypes and therefore in their chemical composition. The first 25 PC scores were used in a supervised

classification model, linear discriminant analysis (LDA), in order to discriminate and classify the data by maximizing the variance between groups. Data reduction by PCA before LDA was

essential because LDA requires that the number of variables is smaller than the number of observations. The smallest number of PC scores was selected for the LDA to prevent data overfitting.

A decreased number of PCs reduced the accuracy of the method in distinguishing the EVs, whereas an increased number did not improve the classification, but progressively decreased accuracy.

Leave-one-out cross-validation was used to test the classification sensitivity, specificity, and accuracy of the LDA model. One-way ANOVA was performed on PC scores to verify that the means

of each group were significantly different, despite within-group variance. Data manipulations and statistical analysis were performed using Origin2017 (v. 9.4, OriginLab, Northampton, MA,

USA). CLS FITTING Reference molecules of some of the major known constituents of EV membrane were used to investigate the lipid content of vesicles. Cholesterol (Chol), ceramide

(N-stearoyl-D-erythro-sphingosine; Cer), sphingomyelin (SM), phosphatidylcholine (16:0/22:6; PCh), L-α-phosphatidylethanolamine (PE), phosphatidic acid (PA), and

monosialotetrahexosylganglioside (GM1) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used to acquire reference spectra using the same acquisition settings as those used

for the EV analysis. Labspec 6 was used for the Classical Least-Squares (CLS) fitting of the PC1 and PC2 spectra, which allows to calculate the contribution of the reference chemicals to the

PC spectra by evaluating any similarities. The resulting coefficients described the relationships between the PC spectra and the reference molecules. DATA AVAILABILITY The datasets

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Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported by the Italian Ministry of Health (Ricerca Corrente 2015, IRCCS Fondazione Don Carlo Gnocchi ONLUS; Ricerca

Corrente RC L1027, IRCCS Galeazzi Orthopaedic Institute) and by the Department of Biomedical, Surgical and Dental Sciences (University of Milan, grant no. 15-63017000-700). AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Nanomedicine and Clinical Biophotonics, IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milano, Italy Alice Gualerzi, Silvia Picciolini, Carlo

Morasso, Renzo Vanna, Marzia Bedoni & Furio Gramatica * Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Università degli Studi di Milano, Milano, Italy Stefania Niada,

 Chiara Giannasi & Anna Teresa Brini * Laboratorio di Applicazioni Biotecnologiche, IRCCS Istituto Ortopedico Galeazzi, Milano, Italy Stefania Niada, Chiara Giannasi & Anna Teresa

Brini * Nanomedicine Center NANOMIB, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy Silvia Picciolini & Massimo Masserini * San Raffaele Telethon Institute

for Gene Therapy (SR-TIGET), Pediatric Immunohematology, San Raffaele Scientific Institute, Milano, Italy Valeria Rossella & Maria Ester Bernardo * Hematology and Bone Marrow

Transplantation Unit, San Raffaele Scientific Institute, Milano, Italy Fabio Ciceri Authors * Alice Gualerzi View author publications You can also search for this author inPubMed Google

Scholar * Stefania Niada View author publications You can also search for this author inPubMed Google Scholar * Chiara Giannasi View author publications You can also search for this author

inPubMed Google Scholar * Silvia Picciolini View author publications You can also search for this author inPubMed Google Scholar * Carlo Morasso View author publications You can also search

for this author inPubMed Google Scholar * Renzo Vanna View author publications You can also search for this author inPubMed Google Scholar * Valeria Rossella View author publications You can

also search for this author inPubMed Google Scholar * Massimo Masserini View author publications You can also search for this author inPubMed Google Scholar * Marzia Bedoni View author

publications You can also search for this author inPubMed Google Scholar * Fabio Ciceri View author publications You can also search for this author inPubMed Google Scholar * Maria Ester

Bernardo View author publications You can also search for this author inPubMed Google Scholar * Anna Teresa Brini View author publications You can also search for this author inPubMed Google

Scholar * Furio Gramatica View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.G. performed Raman experiments; A.G., S.N., G.C. and V.R.

conducted EV isolation and characterisation; A.G., S.P., C.M., R.V., and M.B. analysed and interpreted data; M.M., F.C., F.G., M.E.B. and A.T.B. supervised the work; A.G., C.M., S.P. and

R.V. wrote the original draft manuscript; S.N., G.C., M.E.B., A.T.B., M.M., F.C. and F.G. reviewed and edited the final manuscript. CORRESPONDING AUTHOR Correspondence to Alice Gualerzi.

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spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells. _Sci Rep_ 7, 9820 (2017). https://doi.org/10.1038/s41598-017-10448-1

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