Study design
A pilot trial of nebulization therapy for COVID-19 pneumonia with exosomes of MSCs was performed on seven patients with COVID-19 pneumonia. The study was conducted at Wuxi No. 5 People’s Hospital, China. The safety and scientific validity of this study have been issued in Chinese Clinical Trial Registry (ChiCTR2000030261).
Inclusion criteria
We initially enrolled patients with COVID-19 (age 18–65 years) according to the guidance of the National Health and Health Commission of China [20]. We comprehensively considered the patient's epidemiological history, clinical symptoms, and nucleic acid test results. Informed consent was obtained from all patients participating in this clinical study.
Exclusion criteria
(1) Age < 18 or > 65; (2) severe heart, brain, lung, kidney dysfunction, endocrine disease, haematopoiesis system disease or other serious diseases and psychosis; (3) pregnant and lactating women; (4) participating in other clinical trials or who have participated in other clinical trials in the last 3 months; and (5) unwilling or unable to sign informed consent due to illness.
Patients
The patients were enrolled from February 26, 2020, to April 30, 2020. All enrolled patients were confirmed to have COVID-19 via real-time reverse transcription polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 RNA following the protocol outlined in a previous study [3, 21].
All patients were treated with ritonavir orally, abidol orally, interferon nebulization, or chloroquine phosphate orally (Fig. 1). The clinical, laboratory, and radiological outcomes of all patients were recorded and certified by a trained group of doctors. The detailed records included primary safety data (allergic reactions, secondary infection and life-threatening adverse events) and primary efficacy data (the plasma level of CRP and oxygen saturation). The secondary efficacy outcomes mainly included the total white blood cell count, total lymphocyte count (assessed using a Hitachi 7600-020 automatic biochemical analyser), SARS-CoV-2 nucleic acid detection (assessed by RT-PCR, DAAN GENE Co., Ltd, China), chest CT (assessed using a 320-slice spiral CT scanner, Aquilion One, Toshiba Medical System, Japan), respiratory rate, symptoms (especially fever and shortness of breath), and time of hospitalization. Immune factors were also detected in patient 6 (DxFLEX flow cytometry, BECKMAN, USA).
Umbilical cord processing and cell isolation
An umbilical cord sample was aseptically collected, and the sample was placed in a collection cup containing medium consisting of DMEM with 4,500 mg/mL glucose and antibiotic solution containing 0.1% gentamicin, 0.2% streptomycin and 0.12% penicillin. The sample was placed in a foam box precooled to 4°C with ice and transported to the laboratory within 4 hours of collection.
The sample in the collection cup was rinsed three times with PBS to remove blood clots and then placed in a Petri dish in a biosafety cabinet. The umbilical cord was cut into 5-cm-long pieces using scissors and forceps. The pieces were cut longitudinally to expose the blood vessels and surrounding Wharton’s jelly; the Wharton’s Jelly was scraped away from the blood vessels and placed in a separate Petri dish.
The Wharton’s jelly was cut into 1-mm pieces with scissors, and 5 ml of each piece was added to a T175 culture flask. Then, 15 mL of complete medium (Mesenchymal Stem Cell Basal Medium, Cat# 6114011, Dakewe Bioengineering, China) with 5% cell culture supplement (EliteGro-Adv. GMP, Cat# EPAGMP-500, EliteCell Biomedical, Beltsville, MD, USA) and gently shaken so that the tissue pieces were evenly distributed. The sample was incubated at 37°C in a 5% CO2 incubator for 2 days without disturbing the flasks such that the tissue pieces adhered to the flask.
After 5 days of incubation, the medium was changed, and the appearance of the cells outgrowing from the edge of the explant was assessed on a daily basis; cell outgrowth from the adherent explants was often evident after approximately 7 days of culture. Thereafter, the medium was changed every 3 days or as needed. When cell growth reached 70% confluence, which could be observed at approximately 7 days after the initial cell outgrowth from the explant, subculture of the cells was initiated: the flask was gently shaken so that the tissue pieces dissociated, and they were carefully aspirated together with the supernatant.
The cells remaining in the flask were dissociated by using 4 mL of commercial trypsin solution per flask and incubated at 37°C for two minutes; 5 ml of complete medium was added to neutralize the solution, and the cell suspension was transferred to a centrifuge tube.
The cell suspension was centrifuged at 1,500 x g for six minutes. The pelleted cells were considered passage 0 (P0). For further amplification and characterization, P0 cells were suspended in complete medium, counted, and subcultured at a seeding density of (1.2-1.4) x 104/cm2. Excess cells were cryopreserved as P0 cells.
Characterization of isolated cells
Expression of cell surface markers was analysed using flow cytometry: P5 cells were highly proliferative and positively expressed CD90, CD105, and CD73 but did not express CD34, CD45, CD14, CD19 or HLA-DR. Differentiation potential: P5 cells displayed adipogenic differentiation, chondrogenic differentiation and osteogenic differentiation potentials when cultured in appropriate commercially available differentiation media.
All procedures performed in this study involving human samples were in accordance with the ethical standards of the institutional research committee and the guidelines set by the Declaration of Helsinki.
Isolation and characterization of exosomes isolated from MSCs
Exosomes secreted from MSCs were collected and purified using multiple ultrafiltration steps (Fig. 2a). The secretions were first centrifuged at 4 ℃ at 3000 x g for 20 min and filtered through a 0.22 μm filter to remove any cells or cell debris. The filtered secretomes were then placed in a new sterile EP tube, followed by the addition of 0.2 ml exosome separation and purification solution (Shanghai Gefan Biotechnology Co., Ltd. Product No.: ex010). The contents were mixed well at 4 ℃ overnight and centrifuged at 4 ℃ at 3000 x g for 20 min the following day. After the supernatant was aspirated, it was centrifuged at 4 ℃ for 1500 x g for 5 min to remove the residual solution. After adding 1000 μl sterile PBS to resuspend and precipitate the exosomes, the solution was centrifuged for 1 hour at 100 000 x g using a high-speed centrifuge, and this was repeated three times. The exosome samples were analysed for proper size using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern) and for morphology using transmission electron microscopy (TEM; Tecnai G2 Spirit Bio TWIN). Additionally, successful exosome isolation was confirmed using immunoblotting for known exosome markers. Standard Western blotting was performed using rabbit antibodies against human CD9 (1:2000, ab92726, Abcam), CD81 (1:6000, ab109201, Abcam), and Flotillin 2 (1:6000, ab181988, Abcam). Protein sample loading was monitored by probing the same membrane filter with an anti-β-actin antibody (1:2000, sc-130301, Santa Cruz) (Fig. 2).
After nebulization, the MSC exosomes were sprayed onto a sterile glass plate, and a dish was placed under the glass plate for collecting the liquid containing the exosomes. After nebulization, the size and markers of the MSC exosomes were evaluated again. No significant difference in the size and markers of MSC-derived exosomes before and after nebulization was observed (Fig. 2c and d).
Nebulization of MSC-derived exosomes
After obtaining ethical approval, all patients diagnosed with COVID-19 pneumonia who provided informed consent were treated with nebulization of MSC-derived exosomes. All patients were treated with the same dose of MSCs (1 million cells/kg predicted body weight) according to a previous study [22]. The concentration of exosomes for nebulization for each patient ranged from 7.66e + 0.8 to 7.00e + 0.7 particles/ml based on NanoSight. The exosomes extracted from MSCs were diluted to 5 ml with 0.9% sodium chloride and added to an atomizer (Emedical, Excellentcare Medical Ltd. China). Nebulization was performed twice a day (am 8:30, pm 16:00) for 10 minutes each. The patients were assessed by investigators after receiving the nebulization treatment.
Treatment procedure for MSC-derived exosomes and general patient information
This study was conducted from February 26, 2020, to September 4, 2020. Seven patients diagnosed with COVID-19 pneumonia, including 2 severe cases (patients 2 and 4) and 5 mild cases (patients 1, 3, 5, 6 and 7), were enrolled in the study (Table 1). Patients 1, 2, 3 and 4 received nebulization of MSC-derived exosomes after antiviral treatment for a period of time. Patient 1 had a mild case of COVID-19 and did not have any underlying disease conditions. Patient 2 had a severe case of COVID-19 and liver damage. Patient 3 was a mild case and patient 4 a severe case, both without any underlying disease. Patients 5, 6 and 7 received nebulization of MSC-derived exosomes from the beginning of treatment. Information about all the treatment modalities of the patients was collected. The timepoint of the delivery of MSC-derived exosome nebulization treatment for each patient is shown in Fig 1.
Table 1. General information of the enrolled patients.
Patient 1
|
Patient 2
|
Patient 3
|
Patient 4
|
Patient 5
|
Patient 6
|
Patient 7
|
Sex
|
F
|
M
|
F
|
F
|
M
|
M
|
M
|
Age (years)
|
62
|
53
|
23
|
61
|
43
|
19
|
57
|
Underlying diseases
|
No
|
Liver damage
|
No
|
No
|
Fatty liver
|
No
|
Diabetes mellitus
|
COVID-19 type
|
Common
|
Severe
|
Common
|
Severe
|
Common
|
Common
|
Common
|
Fever (°C, baseline)
|
38.5
|
37.4
|
37.6
|
38.9
|
37.7
|
37.4
|
37.6
|
Cough
|
Yes
|
Yes
|
No
|
Yes
|
No
|
No
|
Yes
|
Weak
|
Yes
|
No
|
No
|
Yes
|
No
|
No
|
No
|
Diarrhoea
|
No
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Shortness of breath
|
No
|
No
|
No
|
Yes
|
No
|
No
|
No
|
Chest tightness
|
Yes
|
Yes
|
No
|
No
|
No
|
No
|
No
|
Oxygen saturation at rest state (%)
|
87
|
74
|
93
|
61
|
97
|
97
|
98
|
Date of diagnosed
|
Feb 9
|
Feb 9
|
Feb 8
|
Jan 31
|
Mar 28
|
Mar 27
|
Aug 20
|
Date of MSCs exosomes treatment
|
Feb 27
|
Feb 27
|
Feb 27
|
Feb 27
|
Apr 1
|
Apr 4
|
Aug 23
|
Date of recovery
|
Mar 2
|
Mar 2
|
Mar 11
|
Mar 9
|
Apr 11
|
Apr 13
|
Sep 4
|
Hospital day
|
22
|
22
|
31
|
38
|
14
|
17
|
15
|
Yes: Presence of relevant clinical symptoms, such as cough, weak, diarrhoea, shortness of breath and chest tightness.
No: No relevant clinical symptoms.
Statistical analysis
Data that were suitable for statistical analysis were analysed using SPSS software (SPSS 22.0). Differences between two groups were assessed using unpaired two-tailed t tests or chi square tests based on the type of data. Data involving more than two groups were assessed using analysis of variance (ANOVA). P values <0.05 indicated statistical significance.