Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Summary

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Abstract

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Introduction

Exosomes (Exo) are a subgroup of extracellular vesicles (EVs), ranging from 30 to 150 nm in size, encapsulating a diverse array of biomolecules, including proteins, nucleic acids, and lipids¹. These biomolecules are derived from the parental cells and are released into the extracellular space. Exo facilitate biomolecular information exchange between cells and their surrounding microenvironment, playing roles in both prokaryotic and eukaryotic systems². The characteristics of exosomes, such as content, size, membrane components, and cellular origin, are highly variable and influenced by the originating cell type, cellular state, and environmental conditions.

Exosomes are commonly classified into three categories based on their source: cell culture-derived, body fluid-derived, and tissue-derived (Ti-Exo). While cell culture-derived exosomes have been extensively studied due to their accessibility and consistent yield, their use is limited by potential alterations in cell characteristics after prolonged culture, which may misrepresent their biological functions^3,4,5. Moreover, most cell cultures are maintained in two-dimensional environments that do not mimic the complex in vivo intercellular interactions, potentially impacting data interpretation⁶. Conversely, exosomes isolated from biological fluids offer a minimally invasive option to monitor disease progression in real time⁷. However, these samples often contain a mixture of exosomes from various origins, complicating the accurate identification of their primary source⁸. Given these challenges, there is an increasing interest in Ti-Exo, which reside in the extracellular interstitium and are key mediators of intercellular signaling.

The spleen plays a crucial role in immune function and maintaining internal homeostasis. Despite existing protocols for isolating Ti-Exo from organs such as the brain, liver, and tumors, practical methods for spleen-derived exosomes are limitedly reported^9,10. This study aimed to establish a practical protocol for isolating exosomes from spleen tissue in mice modified from a previous report¹¹. We detail a method involving collagenase digestion followed by ultracentrifugation, which minimizes the disruption of the cell membrane and ensures high purity and yield of spleen-derived Exo. The characteristics of isolated Exo using this established protocol were validated through electron microscopy (TEM), the nano-flow cytometer (Nano-FCM), and western blot, confirming the protocol's efficacy for further experimental research.

Protocol

The samples were obtained from mice, with ethical approval granted by the Ethics Committee of Guangdong Medical University. Detailed descriptions of materials, equipment, and software used in this protocol are provided in the Table of Materials. The details of the preparation prior to extraction are illustrated in Figure 1, while the process of Spleen-Exo extraction and enrichment is described in Figure 2.

1. Preparation

1. Pre-cool high-speed and ultra-high-speed centrifuges to 4 °C and set the temperature of a desktop shaker to 37 °C.
2. Prepare cell culture dishes (100 mm), 50 mL sterile centrifuge tubes, ice, and sterile cell strainer (70 µm) as shown in Figure 1A, B.
3. Sterilize scissors and forceps using 75% alcohol spray, followed by high-temperature sterilization. These tools are shown in Figure 1C.
4. Place the spleen tissue in a sterile 100 mm Petri dish on ice, and wash off the surface blood with 1x iced sterile phosphate-buffered saline (PBS). Dry the spleen tissues with sterile gauze and weigh them after drying.
   NOTE: If the tissue is frozen, thaw it in a dish for 15 min on ice before proceeding.
5. Moisturize the cell strainer (70 µm) by dropping 1 mL of sterile PBS into the strainer using a sterile transfer pipet.
6. Prepare the digestive buffer (0.1% type I collagenase) using 1x PBS with a vortex mixer.
   NOTE: Use 10 mL of digestive buffer for every 100 mg of spleen tissue. The collagenase concentration was chosen in this study based on the product instructions. For other tissue types, researchers may need to adjust these parameters based on tissue density and composition. Preliminary experiments with varying concentrations and digestion times are recommended to determine the optimal conditions for each tissue type.

2. Tissue digestion

1. Chop the tissue into uniform small pieces with scissors in a culture dish on ice (Figure 3A) and transfer to a 50 mL centrifuge tube with a sterile transfer pipet.
2. Add digestion buffer to the tube according to the spleen tissue weight as indicated above (Figure 3B), then shake the tube with an angle of 45° on a horizontal shaker at 37 °C. Monitor the digestion progress and stop the digestion when tissue chunks have dispersed, and most have lost their shape. Digestion time should vary between tissues. Under the experimental conditions in this study, the digestion time is approximately 30 min.
3. Transfer the digested mixture to a cell strainer (Ф70 µm) at room temperature (RT) to remove fibrous and larger tissue debris.
   NOTE: Complete filtration immediately after digestion.
4. Collect the filtrate in a new 50 mL centrifuge tube.

3. Exosome isolation by differential centrifugation

1. Centrifuge filtrates at 500 x g for 10 min at 4 °C. A pellet containing residual cells, cell debris, and nuclei can be seen after centrifugation, as shown in Figure 3C.
2. Transfer the supernatant to a new tube and centrifuge at 3000 x g for 20 min at 4 °C. A pellet containing apoptotic bodies and microsomes can be seen after centrifugation, as shown in Figure 3D.
3. Transfer the supernatant to a new tube and centrifuge at 10,000 x g for 30 min at 4 °C. Pellet containing microvesicles and plasma membrane can be seen after centrifugation, as shown in Figure 3E.
4. Ultracentrifuge the resulting supernatant at 120,000 x g for 2 h at 4 °C. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3F.
5. Discard the supernatant, wash the pellet with PBS, and ultracentrifuge again at 120,000 x g for 2 h at 4 °C to obtain exosomes. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3G.
6. Dissolve the isolated exosomes with sterile PBS (200 µL per spleen) and store the isolated exosomes in a new 2 mL centrifuge tube at -80 °C.

4. Exosome characterization by transmission electron microscopy (TEM)

NOTE: TEM was used to determine whether the extracted Exo displayed vesicle characteristics. The Exo samples identified by electron microscopy must be fresh samples or stored briefly at 4 °C for a short time.

1. Fix exosomes (2-6 µg/µL) with 2% electron microscopy (EM)-grade paraformaldehyde at RT for 2 h.
2. Place 10 µL of exosome solution on a copper mesh, incubate at RT for 10 min, rinse with sterile distilled water, and blot excess liquid with absorbent paper.
3. Stain the copper mesh with 2% uranyl acetate for 1 min, blot excess fluid with filter paper, and dry under an incandescent lamp for 2 min.
4. Observe the prepared sample under a transmission electron microscope at 80 kV.

5. Size distribution and particle concentration measurement of exosomes

1. Load the standard polystyrene nanoparticles (250 nm) to the nano-flow cytometer.
2. Measure the protein concentration of Exo samples using a BCA protein assay kit. Dilute Exo sample with PBS according to BCA Protein Assay results (dilute the exosomes to 1-10 ng/µL), then load the Exo samples to the nano-flow to measure the side scatter intensity (SSI).
3. Calculate Exo concentration according to the ratio of SSI to particle concentration in the standard polystyrene nanoparticles.
   NOTE: For size measurement, a standard curve is generated by loading standard silica nanoparticles with gradient sizes (68 nm, 91 nm, 113 nm, 155 nm) to the nano-flow cytometer. The loading of the Exo sample follows this. The size distribution of Exo was calculated according to the standard cure.

6. Lysis and immunoblot confirmation of exosome proteins

1. Add 100 µL of radio immunoprecipitation assay (RIPA) lysis buffer to the exosome pellet for protein analysis.
2. Vortex vigorously, cover with parafilm, rock for 20 min at RT, then vortex again.
3. Centrifuge briefly at 1000 x g for 30-60 s to collect the sample, transfer to a new 1.5 mL microcentrifuge tube, and store at -20 °C to -80 °C until analysis.
4. For immunoblot, mix samples with 5x loading buffer to achieve a 1x concentration and prepare for gel electrophoresis.
5. If whole-tissue homogenate controls are desired, use a strong lysis buffer with a protease inhibitor to lyse the tissue, as detailed above. Then, centrifuge the samples at 10,000 x g for 10 min and discard the remaining extracellular matrix, whole cells, or intact cellular debris.
6. Boil the sample buffer containing exosomes or tissue homogenate at 95 °C for 5-10 min. Load equal protein per lane into a 10% SDS-PAGE gel. Load equal protein of tissue homogenate as control.
7. Proceed with gel electrophoresis under a voltage of 80 V for approximately 2 h using the conventional electrophoresis buffer. Perform western blot analysis to confirm Exo protein expression in the purified lysates and compare relative Exo abundance in fractions.

Results

Isolation and purification of spleen-derived exosomes
To isolate exosomes from mouse spleen tissue, we employed a combination of collagenase digestion and differential ultracentrifugation (Figure 2). The initial steps involved pre-treating the spleen tissue to remove surface blood, followed by digestion with Type I collagenase. This enzymatic treatment facilitated the breakdown of extracellular matrix components, thus liberating the exosomes while preserving their integrity. Post-digestion, a series of centrifugation steps were employed to sequentially remove cellular debris, apoptotic bodies, and larger vesicles, culminating in the ultracentrifugation of the supernatant at 120,000 x g to pellet the exosomes. Subsequent isolation procedures yielded PBS-soluble pellets enriched with spleen-derived Exo, as shown in Figure 3G.

TEM results
The morphology of the isolated exosomes was examined using transmission electron microscopy, a gold-standard method to study the characterization of exosomes. TEM is commonly used to validate the presence of exosomes, evaluate the quality of exosomes, and observe exosomes' morphology. The TEM images (Figure 4A) revealed the presence of cup-shaped vesicles, which are characteristic of exosomes. These vesicles displayed a clear lipid bilayer structure, confirming the successful isolation of exosomes from the spleen tissue.

Size distribution of Spleen-Exo
The size distribution and concentration of the exosomes were determined using a nano-flow cytometer. The results (Figure 4B) indicated that the diameter of the isolated exosomes ranged from 30-150 nm, with a peak concentration at approximately 60 nm. The diameter of isolated particles was dominantly from 50-80 nm, with a mean particle concentration of 4.6 x 10⁹ particles/mL. This size range is consistent with previously reported characteristics of exosomes, further validating the isolation protocol.

Western blot analysis
To confirm the presence of exosomal markers, we performed a western blot analysis. The exosomal markers TSG101 and CD9 were detected in the Spleen-Exo samples, whereas GM130, a Golgi matrix protein used as a negative control, was not detected (Figure 4C). In comparison, the protein of these markers was expressed in mouse spleen tissues (Spleen-T). This indicates the high purity of the isolated exosomes, as they were devoid of cellular contaminants.

Figure 1: Supply preparation. (A) 50 mL centrifuge tubes, culture dishes, ice, and surgery tools were placed in a biosafety cabinet. (B) A sterile cell strainer (70 µm). (C) Sterilized surgery tools. Please click here to view a larger version of this figure.

Figure 2: Spleen-Exo isolation procedure. Prepare spleen tissue and cut it into small pieces in iced PBS, followed by digestion with Type I collagenase and centrifugation as indicated. Please click here to view a larger version of this figure.

Figure 3: Representative images of Spleen-Exo isolation. (A) Small spleen blocks ready for subsequent digestion. (B) Spleen tissue blocks with digestion buffer. (C) Pellets after 500 x g centrifugation. (D) Pellets after 3,000 x g centrifugation. (E) Pellets after 10,000 x g centrifugation. (F) Pellets after 120,000 x g centrifugation. (G) Exosome pellets after 120,000 x g centrifugation. Please click here to view a larger version of this figure.

Figure 4: Identification of Spleen-Exo. (A) TEM results showing isolated particles as round or oval membranous vesicles with cup-shaped morphology. Scale bar = 200 nm and 50 nm, respectively. (B) Size distribution analysis by Flow Nano Analyzer showed that the diameter of Spleen-Exo ranged dominantly from 50 nm to 80 nm, with a mean particle concentration of 4.6 × 10⁹ particles/mL. (C) Western blot analysis showing expression of exosome-specific marker proteins (TSG101 and CD9) in Spleen-Exo, with negative expression of GM130. These markers were detectable in protein samples extracted from mouse spleens (Spleen-T group). The representative western blot images were shown (n = 6 tissues). Please click here to view a larger version of this figure.

Discussion

Recent research has reported that spleen-derived exosomes (Spleen-Exo) play a critical role in the treatment of gastric cancer¹². To further elucidate the functions of Spleen-Exo, it is necessary to establish a reproducible and optimal method for their extraction from spleen tissue. While protocols exist for isolating exosomes from brain and liver cancer^13,14, methods for other tissues remain underdeveloped and require further validation. In this study, we have outlined a practical method for isolating Exo from spleen tissue.

Over the past years, various methods have been used to extract Exo in scientific research and applications, including ultracentrifugation, density gradient centrifugation, magnetic bead immunoadsorption, ultrafiltration, and polymer precipitation¹⁵. Ultracentrifugation remains the gold standard method for extracting Exo from various sources. Previous studies have focused on disrupting tissue structures to obtain higher concentrations of Exo, which consists of intracellular and extracellular sources of Exo¹⁶. Collagenase minimizes the impact on the integrity of the cell membrane, ensuring the purity of Ti-Exo³. Collagenase has been used to extract extracellular vesicles from human metastatic melanoma, adipose, colon cancer, and liver cancer tissues^5,10,13,17. However, the concentration and digestion time of collagenase vary among different tissues.

We established a specific protocol for isolating Spleen-Exo using Type I collagenase for digestion, followed by filtration through differential ultracentrifugation, to yield high-quality Spleen-Exo. Ultracentrifugation can effectively remove residual cells, large vesicles, cell debris, and apoptotic bodies from the samples¹⁶. Crescitelli et al. employed collagenase D and gradient ultracentrifugation to isolate small extracellular vesicles with a mean size of 75 nm in diameter⁵. Compared with their protocol, our protocol used type I collagenase and an additional ultracentrifugation step at 120,000 x g to achieve optimal purity of exosomes. In contrast to previous reports using tissue homogenization to obtain tissue suspension^14,18, our approach preserves the membrane integrity of cells in tissues. In addition, Vella et al. applied density gradient centrifugation (a triple sucrose gradient) to generate three groups of particles, which required extra time for the operation¹⁰. This approach produced large vesicles with a size over 200 nm, a heterogeneous population of vesicles with sizes ranging from 50 nm to 200 nm, and small vesicles around 20 nm in size. The protocol described in this study protocol involves a series of ultracentrifugation steps to methodically eliminate residual cells, cell debris, apoptotic bodies, and larger vesicles, thus enhancing the purity of the Exo obtained. We emphasize careful handling during supernatant collection to avoid disturbing the sedimented pellets.

We assess the morphology and purity of the isolated Spleen-Exo through TEM and nanoparticle tracking analysis. Both assays revealed that the spleen-derived particles isolated by this protocol exhibited a lipid bilayer and cup-shaped vesicles with sizes ranging from 30 nm to 150 nm, which are the typical characteristics of exosomes¹⁹. CD9 and TSG101 are exosome markers¹⁹, and GM130, a Golgi protein, is used as a negative marker for exosome identification²⁰. The results showed that Spleen-Exo were positive for CD9 and TSG101, while GM130 was not detected, confirming the successful isolation of high-quality, tissue-derived Exo from spleens.

In achieving high-quality Ti-Exo, selecting the appropriate enzyme type, concentration, and digestion duration, as well as proper tissue storage, is essential. We observed no significant difference in protein concentrations between Exo derived from fresh versus frozen spleen tissues (data not shown), indicating the feasibility of processing large quantities of frozen tissues to reduce the workflow. Isolating Ti-Exo requires a long operating time for ultracentrifuge, making it impractical to complete the whole protocol within an appropriate period when using fresh tissues in clinical applications. Thus, using frozen tissues for exosome isolation would be a better option. Thawing at 37 °C is not suitable for frozen tissues due to incomplete penetration of cryoprotective agents in tissues. It was reported that when performing exosome isolation using frozen tissues thawing on ice, the yield of exosomes showed no significance in content compared with fresh tissues²¹. Thus, we thawed the frozen spleen tissues on ice to perform exosome isolation when using the storage samples at -80 °C.

However, the protocol described in this study has several limitations. The extensive steps involved in extraction and identification are time-consuming. Given the ultimate goal of applying Exo research to clinical disease treatment, faster and more cost-effective methods are becoming increasingly critical. Additionally, while the results are based on mouse tissue samples, the applicability to human tissues remains to be determined.

In conclusion, the protocol provides a method for extracting highly pure Exo from spleen tissue, suitable for detailed downstream analysis. Tissue-derived Exo reflects the microenvironment of their origin more accurately and holds significant potential for elucidating physiological and pathological processes in living organisms. Future studies could adapt this protocol to human tissues, enabling the extraction of clinically relevant exosomes for diagnostic and therapeutic purposes. This technique could be particularly useful in studying the role of exosomes in immune responses, given the spleen's critical function in the immune system. Moreover, the protocol could be modified to isolate exosomes from other tissues, potentially aiding in the development of tissue-specific biomarkers and targeted therapies. By improving our understanding of exosome biology, this protocol can contribute to advancements in precision medicine and the development of novel therapeutic strategies.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70 µm Cell Strainer	Biologix Group Co., Ltd.USA	15-1070
Anti CD9 antibody	Cell Signaling Technology, Inc (CST), USA	983275
Anti GM130 antibody	Proteintech Group, Inc.China	66662-1-lg
Anti TSG101 antibody	Abcam Plc, UK	125011
Cell Culture Dish	Wuxi NEST Biotechnology Co.,Ltd, China	704001
Centrifuge tube	Wuxi NEST Biotechnology Co.,Ltd, China	788211
Collagenase Type I	Sigma-Aldrich Corp., USA	C2674
Desktop Thermostatic Shaker	Shanghai bluepard instruments Co., Ltd., China	THZ-100
Electrophoresis buffer	Wuhan Servicebio Technology Co., Ltd., China	G2081-1L
Enhanced BCA Protein Assay Kit	Shanghai Beyotime Biotechnology Co., Ltd., China	P0010S
Flow NanoAnalyzer	NanoFCM Inc., China	N30E
Fluorescence/Chemiluminescence imaging system	Guangzhou Biolight Biotechnology Co., Ltd., China	GelView 6000Plus
HRP Goat anti-Mouse IgG	Proteintech Group, Inc.China	15014
HRP Goat anti-Rabbit IgG	Proteintech Group, Inc.China	15015
microplate reader	Molecular Devices, USA	CMax Plus
Microscissors	Shanghai Medical Instruments (group) Co., Ltd.,China	WA1010
Microscopic tweezers	Shanghai Medical Instruments (group) Co., Ltd.,China	WA3010
Multifuge X1R Pro centrifuge	Thermo Fisher Scientific, USA	75009750
Ophthalmic scissors	Shanghai Medical Instruments (group) Co., Ltd.,China	Y00030
Ophthalmic tweezers	Shanghai Medical Instruments (Group) Co., Ltd., China	JD1060
Optima XPN-100 Ultrafiltration centrifuge	Beckman Coulter, USA	A94469
phosphate buffered saline (PBS)	Beijing Solarbio Science & Technology Co.,Ltd., China	P1003
RIPA lysis buffer (strong, without inhibitors)	Shanghai Beyotime Biotechnology Co., Ltd., China	P0013K
Ruler	Deli Manufacturing Company, China
SDS-PAGE Sample Loading Buffer, 5x	Shanghai Beyotime Biotechnology Co., Ltd., China	P0015
Transfer Pipet	Biologix Group Co., Ltd.USA	30-0238A1
Transmission Electron Microscope	Hitachi, Japan	H-7650
Ultracentrifugation tube	Beckman Coulter, USA	355618
Western Transfer Buffer	Wuhan Servicebio Technology Co., Ltd., China	G2028-1L