Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Podsumowanie

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Streszczenie

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Wprowadzenie

Osteoarthritis (OA), resulting from joint cartilage and underlying bone breakdown, remains a severe challenge leading to disability^1,2. Without blood and nerve supply, cartilage self-healing ability is minimal once being injured^3,4. In the past decades, therapies based on autologous chondrocyte implantation (ACI) have made some progress in OA treatment⁵. For chondrocyte isolation and expansion, harvesting small cartilage from the OA joint's non-weight bearing area is necessary, causing injuries to the cartilage. Also, the procedure will require a second operation to implant the expanded chondrocytes⁶. Thus, one-step therapies for OA treatment without cartilage injuries are under extensive exploration.

Mesenchymal stem cells (MSCs) have been suggested as promising alternatives for OA treatment^7,8. Originating from multiple tissues, MSCs can differentiate into chondrocytes with specific stimulation. Importantly, MSCs can modulate immune responses via anti-inflammation⁹. Therefore, MSCs hold significant advantages in OA treatment by repairing cartilage defects and modulating the immune response, especially in the inflammation milieu. For OA treatment, MSCs from synovial fluid (SF-MSCs) have recently attracted much attention due to their stronger chondrocyte differentiation ability than other MSC sources^10,11. Notably, at the orthopedic clinic, the extraction of inflammatory SF from the joint cavity is a routine therapy to relieve the pain symptom of OA patients. Extracted inflammatory SF usually is disposed of as medical waste. Both patients and doctors are ready to consider autologous MSCs isolated from the inflammatory SF as OA treatment with very few ethical conflicts. However, SF-MSC therapy is compromised due to tumorigenic risks, long-time storage, and distant shipment barriers.

Exosomes, secreted by many cell types, including MSCs, carry most of the parent cell bio-information. It has been investigated in-depth as a cell-free therapy^12,13. According to the updated resources available on the clinical trial government (ClinicalTrials.gov) website, more extensive exosome clinical studies are initiated and undertaken in the research fields of cancer, hypertension, and neuro-degenerative diseases. SF-MSC exosome treatment could be an exciting and challenging trial to cope with OA. Good manufacturing practice (GMP)-grade and large-scale exosome production are essential for clinical translation. Small-scale exosome isolation has been widely performed based on two-dimensional (2D) cell culture. However, large-scale exosome production strategies need optimization. A large-scale exosome manufacturing method was developed in this study, based on massive SF-MSC culture in xeno-free conditions. After ultracentrifugation from cell culture supernatants, exosome safety and function were validated.

Protokół

This study was approved by the Human Ethics Committee of Shenzhen Second People's Hospital. A schematic diagram of exosomes isolated from hSF-MSCs in vitro protocol is shown in Figure 1.

1. Human SF-MSCs culture and identification

1. Harvest 20 mL of SF using a syringe and needle from clinical OA patients.
   1. Disinfect the knee joint of the OA patient. Puncture from the quadriceps femoris tendon outside the patella into the articular cavity with a 7# needle.
   2. Extract 10 mL of the joint fluid. Cover the puncture site with the transfusion stick and press for 5 min.
2. Discard the SF supernatant after centrifugation at 1,500 x g for 10 min at 4 °C.
3. Resuspend the cell pellet with 10 mL of 1x phosphate buffer saline (PBS), centrifuge at 1,500 x g for 10 min at 4 °C, and discard the PBS.
4. Resuspend the pellet with 10 mL of human MSC culture medium at a cell density of 5 x 10⁴ cells/mL (see Table of Materials), and then plate the suspension in a 100 mm dish.
5. Incubate the dish at 37 °C in an atmosphere containing 5% CO₂.
6. After 48 h, remove the non-adherent cells by changing the medium and wash with 1x PBS. Replace the medium every 3 days for 2 weeks.
7. Collect the cell culture supernatants.
8. Identify SF-MSCs using flow cytometry.
   1. Digest the third generation (P3) of SF-MSCs, centrifuge at 1,000 x g for 5 min at 4 °C. Discard the supernatant and collect the cell pellet.
      NOTE: A passage is recognized as the sub-culture of the cells to another culture dish. The primary cells isolated from SF and seeded on dishes are labeled as passage zero (P0). At about 75% confluence, the cells are digested and detached from the dishes, seeded on other dishes, and labeled as P1.
   2. Add 400 µL of blocking buffer (1% BSA in 1x PBS) to the cell pellet (5 x 10⁴) and allow it to stand for 15 min at room temperature (RT).
   3. Centrifuge at 1,000 x g for 5 min at 4 °C, discard the supernatant, and resuspend the pellet in 100 µL of 1x PBS.
   4. Add 1 µL of CD105, CD73, CD90, CD45, CD34, HLA-DR monoclonal fluorescent antibody (dilution ratio 1:100) (see Table of Materials) per tube and incubate at RT for 30 min.
   5. Wash twice with 1x PBS and discard the supernatant. Collect the cell pellet and resuspend in 100 µL of 1x PBS.
   6. Detect on a flow cytometer up to 10,000 cells using filters of 525/50 and 585/40 to detect the fluorophores.

2. 3D bioreactor cell culture

1. Microcarrier preparation
   1. Swell the dry 0.75 g of microcarriers (see Table of Materials) and hydrate in 1x Dulbecco's Phosphate Buffered Saline (DPBS) (50 mL/g of microcarriers) for at least 3 h at RT.
   2. Decant the supernatant and wash the microcarriers for 5 min in fresh DPBS (50 mL/g of microcarriers). Discard the PBS and replace it with fresh 1x DPBS (50 mL/g of microcarriers).
   3. Sterilize the microcarriers by autoclaving (121 °C, 15 min, 15 psi). Allow the sterilized microcarriers to settle, decant the supernatant.
   4. Briefly rinse the microcarriers in the culture medium (50 mL/g of microcarriers) at RT. Allow the microcarriers to settle, discard the supernatant.
2. Perfusion bioreactor
   1. Sterilize the bioreactor by autoclaving (121 °C, 15 min, 15 psi).
   2. Count the number of SF-MSCs and allocate 2.5 x 10⁷ SF-MSCs and microcarriers to the bioreactor perfused with GMP-grade MSC culture medium (250 mL).
   3. Put the bioreactor in an incubator with 5% CO₂ at 37 °C. Rotate the bioreactor at a speed of 15 rpm. Change the culture medium every 6 days.
   4. Collect the cell culture supernatants and microcarriers for further analysis after culture for 14 days.

3. Exosome identification and safety detection

1. Ultracentrifugation
   1. Centrifuge the cell culture supernatant at 300 x g for 10 min at 4 °C and collect the supernatant; discard the cellular debris.
   2. Centrifuge the supernatants at 2,000 x g for 10 min at 4 °C and collect the supernatant; discard the larger vesicles (apoptotic bodies and some larger microvesicles).
   3. Centrifuge the supernatant again at 10,000 x g for 30 min at 4 °C to remove larger vesicles; collect the pellets and resuspend in 40 mL of 1x PBS.
   4. Centrifuge the resuspended pellets at 120,000 x g for 70 min at 4 °C, discard the supernatant, and resuspend the pellets that contain exosomes in 500 µL of 1x PBS.
2. Nanoparticle tracking analysis (NTA)
   NOTE: For each run, 500 µL of the sample were injected into the chamber at a flow rate of 30 µL/min. Perform the analysis at 24.4 °C-24.5 °C.
   1. Dilute the freshly isolated exosome samples with sterile 1x PBS to 1 mL containing 10⁷-10⁹ /mL of particles and inject them into the nanoparticle tracking analysis system (see Table of Materials).
   2. Manually set the capture and analysis system according to the manufacturer's protocol. Visualize the particles by laser light scattering and capture their Brownian motion on digital video.
   3. Analyze the recorded videotapes utilizing software (see Table of Materials) based on tracking at least 200 individual particles per run.
3. Transmission electron microscopy
   1. Fix the exosomes in 4% paraformaldehyde (in cold 1x DPBS) for 5 min.
   2. Mount 5 µL of the exosomes on copper grids. In this experiment, the concentration of exosomes is 1 mg/mL quantified by a protein assay kit (see Table of Materials).
   3. Embed the exosomes in 3% phosphotungstic acid for 10 min on ice. Remove the excess acid and dry the samples at RT.
   4. Image the exosome samples by a TEM at an acceleration voltageof 100 kV.
4. Western blotting
   1. Add 300 µL of lysis buffer (1% Triton X-100, 0.1% SDS, 0.1 M Tris HCl, pH 7) and protease inhibitors cocktail (see Table of Materials) to the exosomes.
   2. Mix the exosomes in the lysis buffer by pipetting up and down and allow it to stand on ice for 20 min.
   3. Centrifuge the mixture at 9,391 x g for 10 min at 4 °C and collect the supernatant. Measure the protein concentration using a protein assay kit (see Table of Materials).
   4. Add 100 µL of 4x protein loading buffer and heat at 100 °C for 10 min.
   5. Load 15 µL of proteins at a concentration of 10 mg/mL and run by gel electrophoresis (120 V, 70 min) and electroblotting at 100 V, 60 min at 4 °C.
   6. Detect the non-exosome-specific markers (calnexin) and exosomal biomarkers (CD9, CD63, and CD81) by fluorescent western blotting (see Table of Materials).
5. Safety test
   1. For bacteria, fungi, and Mycobacterium tuberculosis detection follow steps 3.5.2-3.5.5.
   2. Seed 100 µL of the exosome solution (1 mg/mL) on a blood agar culture plate (see Table of Materials) and incubate the culture plate at 37 °C for 24 h.
   3. Seed 100 µL of the exosome solution (1 mg/mL) on a sabouraud agar medium plate (see Table of Materials) and incubate at 35 °C for 48 h.
   4. Seed 100 µL of the exosome solution (1 mg/mL) on a Lowenstein-Jensen culture medium plate (see Table of Materials) and incubate at 37 °C for 3 weeks.
   5. Detect the appearance of bacteria, fungi, and Mycobacterium tuberculosis colonies by macroscopic observation.
      NOTE: Criteria for evaluating the safety of exosomes are the absence of microorganisms on the culture plate.
   6. For Mycoplasma detection, follow steps 3.5.7-3.5.9.
   7. Resuspend the exosomes in 50 µL of buffer solution included in the kit and heat at 95 °C for 3 min.
   8. Amplify the Mycoplasma in exosome solution using a PCR Mycoplasma detection kit (see Table of Materials).
   9. Detect the PCR products on 1.5% agarose gel electrophoresis (30 min, 120 V).

4. In vitro exosome function detection

1. Exosome labeling
   1. Label 100 µL of exosomes (1 mg/mL) with 1 mM Dil (1000x) (see Table of Materials) and incubate at RT for 30 min.
   2. Recover the exosomes by ultracentrifugation at 100,000 x g for 70 min. Resuspend the pellet in 500 µL of 1x PBS.
2. Loading of Cy3-miR-140 mimics into exosomes
   1. Mix 1 µg/µL of exosomal protein and 5 µmol/mL of Cy3-miR-140 in a final volume of 400 µL of electroporation buffer (1.15 mM potassium phosphate (pH 7.2), 25 mM potassium chloride and 21% (v/v) (see Table of Materials).
   2. Transfer the mixture into cold 0.4 cm electroporation cuvettes and electroporate at 300 V/100 µF capacitance using a gene transfection system (see Table of Materials).
   3. Immediately after electroporation, maintain the mixture at RT for 30 min to ensure the exosome membrane is fully restored.
   4. Treat the mixtures with one unit of RNase A (see Table of Materials) for 20 min to eliminate the miRNA mimics outside the exosomes.
   5. Ultracentrifugate the exosome mixture at 120,000 x g for 90 min, discard the supernatant and resuspend the exosomes in 500 µL of 1x PBS.
3. Exosome uptake assay
   1. Seed the chondrocytes (1 x 10⁷) in 35 mm confocal dishes with 1 mL of DMEM/F12 containing 10% FBS.
   2. After 24 h, treat the chondrocytes with DiI-labeled exosomes (100 ng/mL) or cy3-miR-140 loaded exosomes (100 ng/mL) for 1 h.
   3. Wash with 1x PBS three times, and then label with DAPI (10 ng/mL) for 10 min at RT.
   4. Record the fluorescence signal in confocal laser scanning fluorescence microscopy (CLSM) using the appropriate filters (see Table of Materials).

5. Statistical analysis

1. Perform statistical analysis using appropriate statistical software.
   NOTE: Representative data in each figure were expressed as the mean ± SD. The Student's t-test was used for the comparison of two groups using statistics software. A one-way ANOVA followed by Tukey's multiple was performed in the case of comparisons among multiple groups. P values <0.05 (_*), <0.01 (_**), or <0.001 (_***).

Wyniki

Flow cytometry was used to identify the surface markers of SF-MSCs, according to the minimal criteria to define human MSCs recommended by the International Society for Cellular Therapy^14,15. Flow cytometry analysis revealed that SF-MSCs cultured in this study met the identification criteria of MSCs. They were negative for CD34, CD45, and HLA-DR (below 3%) and positive for CD73, CD90, and CD105 (above 95%) (Figure 2).

Dyskusje

The mesenchymal stem cells have been widely used in regenerative medicine due to their self-renewal, differentiated into tissue cells with specialized functions, and paracrine effects^16,17. Notably, the paracrine effects exerted by exosomes have attracted much attention¹⁸. Exosomes carry the bio-information of MSCs and perform their biological function and overcome MSC shortcomings, such as troublesome storage and shipment. Thus, exosomes ...

Materiały

Name	Company	Catalog Number	Comments
BCA assay kit	ThermoFisher	23227	Protein concentration assay
Blood agar plate	Nanjing Yiji Biochemical Technology Co. , Ltd.	P0903	Bacteria culture
CD105 antibody	Elabscience	E-AB-F1243C	Flow cytometry
CD34 antibody	Elabscience	E-AB-F1143C	Flow cytometry
CD45 antibody	BD Bioscience	555483	Flow cytometry
CD63 antibody	Abclonal	A5271	Western blotting
CD73 antibody	Elabscience	E-AB-F1242C	Flow cytometry
CD81 antibody	ABclonal	A5270	Western blotting
CD9 antibody	Abclonal	A1703	Western blotting
CD90 antibody	Elabscience	E-AB-F1167C	Flow cytometry
Centrifuge	Eppendorf	Centrifuge 5810R
CO2 incubator	Thermo		Cell culture
Confocal laser scanning fluorescence microscopy	ZEISS	LSM 800
Cytodex	GE Healthcare		Microcarrier
Dil	ThermoFisher	D1556	Exosome label
EZ-PCR Mycoplasma detection kit	BI	20-700-20	Mycoplasma detection
Flowcytometry	Beckman		MSC identification
Gene Pulser II System	Bio-Rad Laboratories	1652660	Gene transfection
GraphPad Prism 8.0.2	GraphPad Software, Inc.	Version 8.0.2
HLA-DR antibody	Elabscience	E-AB-F1111C	Flow cytometry
Lowenstein-Jensen culture medium	Nanjing Yiji Biochemical Technology Co. , Ltd.	T0573	Mycobacterium tuberculosis culture
MesenGro	StemRD	MGro-500	MSC culture
Nanosight NS300	Malvern	Nanosight NS300	Nanoparticle tracking analysis
NTA 2.3 software	Malvern		Data analysis
Odyssey FC	Gene Company Limited		Fluorescent western blotting
OptiPrep electroporation buffer	Sigma	D3911	Gene transfection
Protease inhibitors cocktail	Sigma	P8340	Proteinase inhibitor
RNase A	Qiagen	158924	Removal of RNA
Sabouraud agar plate	Nanjing Yiji Biochemical Technology Co., Ltd.	P0919	Fungi culture
TEM		JEM-1200EX
The Rotary Cell Culture System (RCCS)	Synthecon	RCCS-4HD	3D culture
Ultracentrifuge	Beckman	Optima XPN-100	Exosome centrifuge