MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH HANOI MEDICAL UNIVERSITY ====== VU THI THU TRANG APPLICATION OF AUTOLOGOUS BONE MARROW STEM CELLS THERAPY IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE[.]
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEATH HANOI MEDICAL UNIVERSITY ====== VU THI THU TRANG APPLICATION OF AUTOLOGOUS BONE MARROW STEM CELLS THERAPY IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE MANAGEMENT Specialism : Pulmonary Medicine Code : 9720107 ABSTRACT OF THESIS HA NOI – 2023 The thesis has been completed at HANOI MEDICAL UNIVERSITY Supervisors: A Prof Phan Thu Phuong MD PhD Reviewer 1: A Prof Nguyen Huy Luc MD PhD Reviewer 2: A Prof Bach Khanh Hoa MD PhD Reviewer 3: A Prof Nguyen Viet Nhung MD PhD The thesis will be present in front of board of university examiner and reviewer lever at… on … This thesis can be found at: National Library National Medical Informatics Library Library of Hanoi Medical University 2023 THE LIST OF WORKS HAS PUBLISHED AND RELATED TO THE THESIS Ngô Quý Châu, Phan Thu Phương, Nguyễn Tuấn Tùng, Vũ Văn Trường, Vũ Thị Thu Trang*, Nguyễn Thanh Thủy, Đào Ngọc Phú, Đặng Thành Đô, Nguyễn Đức Nghĩa (2019) “Kết bước đầu ghép tế bào gốc tự thân từ tuỷ xương điều trị bệnh phổi tắc nghẽn mạn tính Trung tâm Hơ hấp, Bệnh viện Bạch Mai” Tạp chí Y học Việt Nam, tập 477 số đặc biệt tháng năm 2019, tr30-38 * Tác giả chịu trách nhiệm Vu Thi Thu Trang, Phan Thu Phuong, Nguyen Tuan Tung, Vu Van Truong, Nguyen Huy Binh, Pham Cam Phuong, Nguyen Thanh Thuy, Dao Ngoc Phu, Dang Thanh Do, Nguyen Duc Nghia, Vu Van Giap, Ngo Quy Chau (2020) “Clinical effects of autologous bone marrow derived stem cell therapy for patients with chronic obstructive pulmonary disease at Bach Mai hospital” Journal Of Medical Research 136 (12) – 2020: p99-109 P Thu Phan, T.T Thu Vu*, G Van Vu, T Tuan Nguyen, T Van Vu, P Cam Pham, T Thanh Nguyen, B Huy Nguyen, K Ba Nguyen, Q Ngoc Tran, T Huyen Tran, H Quoc Phan, P Ngoc Dao, N Duc Nguyen, D Thanh Dang, H Thi Chu, K Trong Mai, C Quy Ngo (2021).“Quality and safety of autologous bone marrow derived stem cell separation procedure in chronic obstructive pulmonary disease patients” Journal of functional ventilation and pulmonology, Issue 36 Volume 12, 4/2021, p1-6 * Correspondent author INTRODUCTION Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide Although the disease progresses slowly, is chronic in nature and does not cause death at an early stage, the consequences caused by the disease to the patients, families and society are very severe In the advanced stages of the disease, the patient becomes disabled, severely impaired in health and quality of life, even with treatment Today, the drugs to treat the disease are increasingly optimized, but they not work to prevent the pathogenesis of COPD The disease continues to progress to severe stages, eventually to death Stem cells with characteristics such as high differentiation ability, immunoregulatory ability, tissue regeneration are expected to bring benefits to patients and improve quality of life In Vietnam as well as in the world, the number of studies applying stem cells therapy in general and bone marrow (BM) stem cells in particular in the treatment of COPD is still small and the follow-up time is limited In addition, autologous BM stem cell sources have been studied in normal people and in the treatment of some diseases, but there have been no studies evaluating the characteristics of bone marrow stem cells in patients with COPD Therefore, we conducted a study on the topic "Application of autologous bone marrow stem cells in chronic obstructive pulmonary disease management" with two objectives: Characterization of bone marrow fluid and autologous bone marrow stem cell grafts of patients with COPD Outcomes of application of autologous BM stem cells therapy in COPD management Necessity of the thesis: The prevalence of moderate and severe COPD in Vietnam ranks highest in the Asia Pacific region, accounting for 6.7% of the population In the near future, the burden of COPD will still tend to increase, due to continued exposure to risk factors and an aging population Current measures to treat COPD include: stopping exposure to risk factors, using drugs (bronchodilators, corticosteroids, phosphodiesterase inhibitors ), pulmonary rehabilitation, oxygen support, mechanical ventilation etc However, despite comprehensive and optimized treatment, the disease continues to progress over time Therefore, an urgent need today is to find new treatments with the ambition of stopping or even reversing the course of the disease In recent years in the world, there have been many studies on the use of stem cells in the treatment of various diseases including respiratory diseases such as pulmonary fibrosis, COPD However, the number of studies and follow-up time of clinical trials on stem cells therapy are still limited, there are many questions about the safety and mechanism of action of exogenous cells after being introduced into chronic damaged lungs still needs to be answered New contribution of the thesis: This is the first study to apply autologous BM stem cell therapy in the treatment of COPD, conducted in Vietnam The study results provide indicators on the characteristics of bone marrow fluid, nucleated and mononuclear cell composition in the bone marrow fluid of patients with COPD as well as characteristics of autologous bone marrow stem cell graft of patients with COPD The study demonstrates the safety of autologous stem cell therapy from bone marrow for COPD treatment, as well as the initial results of the therapy when treating COPD patients The thesis also provides scientific data as a basis for further studies Thesis outline: The thesis consists of 119 pages, covering: introduction (2 pages), overview (39 pages), object and method of research (21 pages), results (27 pages), discussion (28 pages), conclusion (2 pages) It has 48 tables, figures, charts 154 References, including English and Vietnamese versions CHAPTER 1: OVERVIEW 1.1 Overview of COPD COPD definition According to the definition of the Global Initiative on COPD (GOLD), COPD is a common preventable and treatable disease, characterized by chronic airflow obstruction, often progressive with a progressive response Excessive chronic inflammatory response in the respiratory tract and lung parenchyma to noxious particles or gases COPD pathogenesis The pathogenesis of COPD is very complex, in which inflammatory factors play a central role The hallmark of COPD is frequent, excessive inflammation of the entire airways and lung parenchyma This inflammatory reaction is much more amplified than the usual inflammatory responses of the airways Cells involved in the inflammatory response release a variety of inflammatory mediators that chemotaxis other inflammatory cells and proinflammatory mediators that amplify inflammation and promote tissue remodeling, change the normal structure and function of the lung Imbalance of Proteinase - Proteinase inhibitors is the most important pathogenesis of the development of pulmonary emphysema and loss of lung elasticity In COPD the balance is tilted towards increased proteolysis or increased proteinase Oxidative activation not only damages lung tissue but also participates in the imbalance of protease - anti-protease Antioxidants also support inflammation by promoting the activity of genes that produce inflammatory chemical mediators such as IL-8, TNF- COPD diagnosis Thoroughly explore the history of exposure to risk factors for the disease, perform a clinical examination to find diagnostic-oriented signs such as a history of exposure to risk factors (smoking, dust smoke), history of cough , chronic sputum production or persistent dyspnea, increasing with exertion Spirometry is the gold standard for diagnosing and assessing the degree of airway obstruction in patients with COPD Diagnosis is confirmed when the obstructive disorder does not completely recover after the bronchodilator test: the FEV1/FVC index < 70% after the test 1.2 Stem cells and sources of stem cells Definition Stem cells are unspecialized cells that can self-renewal and can also differentiate into more mature cells with specialized functions In humans, stem cells have been found in early embryos; in some fetal tissues, umbilical cord and placenta; and in some mature organs Sources of stem cells: embryonic stem cells, fetal stem cells, adult stem cells Bone marrow stem cells Bone marrow is the home of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial stem/progenitor cells (EPCs), and a few other rare types of stem cells In which, HSC and MSC have been studied and applied widely Bone marrow MSCs Definition of MSC embryo, which originates mainly in the mesoderm and makes up the majority of connective tissue cells in the adult body Mesenchymal Stem Cells (MSCs) are multipotent stromal cells that can differentiate into many different cell types of connective tissue including osteoblasts, chondrocytes, muscle cells, and stromal cells fat cells, etc Criterias of MSC The International Society for Cell Therapeutics (ISCT) Committee on Tissue and MSCs has proposed a set of standards for this cell type, including: MSC must be plastic-adherent when maintained in standard culture conditions MSC must express CD105, CD73 and CD90, and lack expression of CD45, -DR surface molecules MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro BM MSCs characteristics Human MSCs are usually isolated from the mononuclear cell layer of the bone marrow Besides regenerative potential, MSCs have been shown to have antiinflammatory, immunomodulatory properties The anti-inflammatory activity of MSCs can be attributed to the reduction of lymphocyte proliferation, natural killer and dendritic cells, and related chemical mediators: TGFIL-10 and NO MSCs have immunosuppressive ability, modulating the function of T cells and B cells MSC also has the ability to modulate immunity through the effect of reducing the maturation and function of dendritic cells, inhibiting dendritic cells proliferation, differentiation and chemotaxis of B cells in vitro Producing BM stem cells graft protocol Collection of BM stem cells BM stem cells extraction: manually or automatically Stem cells preservation Fresh stem cell grafts After collection, stem cells are stored at 4°C for a maximum of 72 hours and then infuse to the patient This method is usually applied to facilities that not have sufficient conditions to be able to store stem cells for a long time Cryopreserved stem cell grafts After being collected, if the storage period is longer than days, it is frozenized and stored at -80oC or lower The stem cells grafts were stored for a long time at subzero temperatures (-196oC), in liquid nitrogen Thawing and infusion of stem cells to the patient Evaluation and quality control of stem cell grafts Nucleated cells count Count the percentage of viable cells by Trypan Blue staining method Cluster culture Identification of live CD34+ and MSC cells by flow cytometry Determine the state of infection 1.3 Application of autologous BM stem cells in COPD management 1.3.1 Pre-clinical trials In general, the studies showing the effectiveness of the treatment of COPD in experimental animals are mainly attributed to the chemical mediators secreted by MSCs These substances reduce the inflammatory response, modulate the immune response, improve epithelial and endothelial cell permeability, and promote tissue damage repair In addition, some pneumocytes are regenerated, which can also contribute to amelioration of the disease These preclinical studies demonstrate the therapeutic potential of MSCs with COPD in humans 1.3.2 Clinical trials applying BM stem cells in COPD Figure 0.1 Clinical trials applying BM stem cells in COPD Trial Weiss cs Phase Stem cells Number Results Allogeneic BM 62 COPD No toxicity or serious MSC vs placebo patients, events or deaths Trial Phase Stem cells Number 1x108cells/1 moderate infusion and severe Once a month in months Results There was no difference in lung function or quality of life compared with the control group Decrease in CRP month after infusion Ribeiro-Paes Autologous BM severe No side effects cs MSC 1x108 COPD Little improvement in cells/kg, single patients respiratory function, infusion little improvement in health status and quality of life Stolk cs Autologous BM COPD No side effects MSC 1- patients, Alveolar membrane 2x106cells/kg severe and increases CD31 cell Twice, week much expression apart severe 1.3.3 Clinical trials applying stem cells in COPD in Vietnam In 2015, Le Thi Bich Phuong et al conducted a pilot study, without a control group at Van Hanh Hospital (Ho Chi Minh City), Military Hospital 103, National Lung Hospital, initially evaluate the role of mesenchymal stem cells from allogeneic umbilical cord in the treatment of COPD in 20 patients Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were collected from umbilical cord samples from donors at birth Purified UC-MSCs were obtained through multiple stages and qualified for a one-time intravenous infusion to study patients Study results showed that no infusion-related toxicity, death, or serious adverse events occurred that were considered related to the use of UC-MSC Patients receiving UC-MSC infusion showed a significant reduction in mMRC, CAT scores and exacerbations The mMRC value dropped sharply from before treatment to after 1, and months of treatment Similarly, CAT scores also decreased significantly from 10.05 before treatment to 6.5 at month after treatment, 4.0 at months after treatment and 2.0 at months after treatment COPD exacerbations significantly decreased from before treatment to at months after treatment However, FEV1, CRP and 6-minute walking test were not significantly improved after treatment (1, and months) compared with before treatment Some other medical facilities also initially conducted TBG treatment for a number of diseases, including COPD However, to date, no other authors have published research results on TBG therapy for COPD CHAPTER METHODOLOGY 2.1 Subjectives 60 moderate and severe COPD patients at Respiratory Center, Bach Mai hospital 2.1.1 Inclusions criteria The patient must meet all of the following criteria: - Male and female patients, between the ages of 40 and 80 - Patient was diagnosed with COPD according to GOLD 2016 - Patient was diagnosed with COPD after 40 years of age - Having at least exacerbations or exacerbation requiring hospitalization in the previous year - Agree to be treated with stem cell transplant and sign an undertaking to accept possible complications before doing any intervention during treatment 2.1.2 Exclusion criteria Patients with any of the following criteria were not included in the study: - Having other lung diseases (asthma, active tuberculosis, restrictive lung disease, idiopathic pulmonary fibrosis, lung cancer, pneumococcal disease ) - Weight < 40 kg - Have a bacterial or viral infection - Had an exacerbation of COPD requiring hospitalization within the previous weeks - Current or recent quit smoking within months - Are breastfeeding, pregnant or planning to become pregnant - Unstable or life-threatening cardiovascular disease: o Heart failure with left ventricular ejection fraction < 40% o Myocardial infarction or unstable chest pain in the past months o Heart valve disease, cardiomyopathy, congenital heart disease o Severe arrhythmia 10 at the bone marrow aspiration site, infection at the aspiration site bone marrow and waiting for a stem cell transplant - The obtained stem cells graft is sampled for quality assessment tests, the rest will be divided into parts: part is used for the 1st infusion, the rest is stored in liquid nitrogen The preserved portion will be thawed and given to the patient at months after the first infusion - Autologous stem cell graft from bone marrow was evaluated at the time of the 1st and 2nd infusion + Determine the number of cells by automatic counter + Determine the percentage of viable cells by Trypan Blue + Determine the percentage of CD34 + cells and the surface marker panel of MSCs (CD90+, CD105+, CD73+) flow cytometry technique + Culture of bacteria, fungi for each stem cell graft The criteria for autologous BM stem cell production: + Viable cells bef + The number of nucleated cells is at least 300 x 106 cells + Proportion of stem cells with markers (CD90+, CD73+, CD105+) and stem cells with CD34+ immune phenotype + Culture of bacteria and fungi with stem cells to determine sterility - Perform the first autologous BM stem cell infusion after extraction for the patient After months, the patient is examined and tested as well as evaluated as in step and step If the patient meets the criteria of the indication and has no contraindications, they will receive a second infusion of stem cells with thawed stem cells after storage in liquid nitrogen - Patients are closely monitored during stem cells infusion and immediately after TBG infusion (1st and 2nd time) Long term follow up - Patients in both groups were maintained on the basic treatment regimen of COPD according to GOLD guidelines to maintain the stability of the disease and drugs being treated for other medical conditions not in the exclusion criteria such as: : hypertension, diabetes, The total follow-up time of patients in both groups was 12 months after the first stem cell infusion or from the time of participating in the study with patients in the control group, followed up once a month 11 2.4 Variables - Characteristics of bone marrow fluid: volume, cell count, mononuclear cells - Characteristics of bone marrow stem cells: + Percentage of viable cells in the pre-transplant stem cell mass The number of nucleated and mononuclear cells in the stem cell mass + Number and percentage of cells with immune phenotype (CD90+, CD73+, CD105+) and immune phenotype CD34+ + Culture of stem cells for bacteria and fungi before and after storage - Rate of undesirable effects, complications related to the procedure - Rate of COPD exacerbations, mortality rate after 12 months of follow-up - Clinical features: mMRC, SGRQ, CAT, 6MWD - Subclinical results: Pulmonary function test + CT scan before and after intervention, quantitative analysis + CRP, IL-6, IL-8, IL-10, IL- - + Arterial blood gas 2.5 Statistical methods - The collected data were processed using SPSS 20.0 software 2.6 Ethical issues The study is a branch of a state-level research project, approved by the Bach Mai Hospital Ethics Committee and the Biomedical Research Ethics Board of the Ministry of Health CHAPTER RESULTS 3.1 Baseline characteristics of patients Table 0.1 Baseline characteristics of patients Index Year, (SD) Sex, male, n (%) Pack-year, (SD) 6MWD (m), FVC (Lít), FVC%, (SD) (SD) (SD) Intervention (n=30) 64,30 (7,87) Placebo (n=30) 64,33 (5,93) p 0,985 30 (100%) 30 (100%) 26,58 (12,14) 26,03 (13,02) 0,866 362,27 (89,31) 371,64 (67,46) 0,635 1,98 (0,42) 2,01 (0,52) 0,801 60,10 (11,42) 60,87 (14,95) 0,824 12 FEV1 (Lít), FEV1%, CAT, (SD) (SD) (SD) SGRQ, (SD) 0,89 (0,28) 0,92 (0,31) 0,681 35,93 (9,35) 37,77 (12,42) 0,585 25 (83,3) 29 (96,7) 0,195 23,67 (5,33) 22,33 (6,30) 0,380 54,66 (13,21) 60,24 (18,46) 0,183 3.2 Characteristics of bone marrow stem cells in COPD patients 3.2.1 BM fluid collection Table 0.2 Characteristics of the 1st ml of BM fluid (n=30) Index Mean (SD) Median Min Max Nucleate cell counts (G/L) 63,15 (43,69) 59,52 7,68 237,50 WBC (G/L) 50,52 (34,95) 47,62 6,14 190,00 Rate of mononuclear cell (%) 20,62 (7,72) 18,00 11,00 45,00 Mononuclear cell counts (G/L) 12,00 (7,69) 10,32 3,36 38,00 Table 0.3 Characteristics of pre-extraction BM fluid (n=30) Index Mean (SD) Min Max 340,43 (43,43) 270 409 Nucleate cell counts (G/L) 21,47 (6,34) 12,08 33,35 Rate of mononuclear cell (%) 26,28 (7,86) 11,00 43,10 Mononuclear cell counts (G/L) 5,68 (2,07) 2,53 10,68 RBC (T/l) 3,78 (0,41) 3,20 5,06 Hb (g/l) 109,8 (8,69) 94 128 Volume (ml) 3.2.2 BM fluid extractions Table 0.4 Characteristics of extracted BM stem cells graft Index Mean (SD) Volume (ml) Min Max 59,75 (23,31) 26,60 98,99 4931,73 (1883,40) 2181,2 8117,18 26,57 (7,29) 15,00 42,00 1255,10 (521,68) 552,69 2700,85 97,43 (2,50) 90 99 82 Nucleated cell counts (G/L) Total nucleated cells (10 ) Rate of mononuclear cells % Total mononuclear cells (10 ) Viable mononuclear cell rate (%) 13 Total CD34 (x 106) CD34 cell count (cells/µl) 31,64 (27,50) 3,59 139,51 383,67 (336,43) 43,78 1701,32 Rate CD34/CD45 (%) 0,97 (0,58) 0,25 3,30 CD 34/kg (103 cells/kg) 593,17 (505,77) 78,04 2632,23 Viable CD 34 rate (%) 78,52 (18,33) 32,95 98,06 1,005 (0,792) 0,156 0,325 Total MSC (x 10 cells) MSC counts (cells /µl) 12,22 (9,70) 1,91 39,70 MSC/nucleated cells (%) 0,040 (0,084) 0,0027 0,3980 MSC/kg (103 cells/kg) 18,84 (15,52) 3,72 58,13 Viable MSC rate (%) 86,56 (13,31) 53,70 st 100,00 nd Table 0.5 Characteristics of BM stem cells graft for the -2 infusion Index Volume (ml) Viable nucleated 1st stem cells graft (n=30) ± SD (Min; Max) 2nd stem cells graft (n=30) ± SD (Min; Max) 39 47,5 97,43±2,50 81,90±5,99 (90,00; 99,00) (75,00; 95,00) 59,75±23,31 37,13±13,04 (26,60; 98,99) (17,10; 59,25) 15,01±13,06 10,89±6,98 (1,71; 66,35) (2,28; 28,79) 383,67±336,43 228,07±146,97 (43,78; 1701,32) (48,00; 606,00) 0,97±0,58 0,75±0,37 (%) (0,25; 3,30) (0,33; 2,03) Viable CD 34 rate 78,52±18,33 83,41±8,47 (32,95; 98,06) (57,29; 95,37) 0,473±0,390 0,280±0,256 (0,075; 1,509) (0,095; 1,268) cell rate (%) Nucleated cell counts (G/L) Total CD34 (x 106) CD34 cell count (cells/µl) Rate CD34/CD45 (%) Total MSC (x 106 cells) MSC counts (cells /µl) 12,22±9,70 5,85±5,37 (1,91; 39,70) (2,00; 26,70) 14 Index 1st stem cells graft (n=30) ± SD (Min; Max) 2nd stem cells graft (n=30) ± SD (Min; Max) MSC/nucleated cells 0,040±0,084 0,01437±0,0133 (0,0027; 0,3980) (0,005; 0,057) 86,56±13,31 77,64±12,99 (53,70; 100) (50,32; 99,00) (%) Viable MSC rate (%) BM stem cells graft microbiological culture: 100% negative 3.3 Application of BM stem cells in COPD management Table 0.6 Clinical index before and after treatment Index CAT (SD) Baseline (T0) (n = 30) Results months after 1st infusion (T6 L1) (n = 30) Results p 23,67 21,27 (5,33) (3,82) 25 20 n (%) (83,33) (66,67) 6MWD (mét) 362,27 428,47 (89,31) (82,86) 54,66 47,14 (13,21) (10,55) (SD) SGRQ (SD) 0,007 0,150 0,000 0,000 Baseline (T0) (n = 29) Results months after 2nd infusion (T6 L2) (n = 29) Results p 23,55 19,14 (5,38) (2,94) 24 19 (82,76) (65,52) 363,56 454,14 (90,61) (104,54) 54,52 44,05 (13,42) (9,64) 0,000 0,180 0,000 0,000 Table 0.7 Spirometry before and after treatment Index FVC (L) FVC % Baseline (T0) (n = 30) Results months after 1st infusion (T6 L1) (n = 30) Results p 1,98 2,08 (0,42) (0,43) 60,10 63,9 0,177 0,075 Baseline (T0) (n = 29) Results months after 2nd infusion (T6 L2) (n = 29) Results p 1,98 2,19 (0,42) (0,50) 59,69 67,34 0,000 0,000 15 Baseline (T0) (n = 30) Results Index (11,42) 0,89 FEV1 (L) (0,28) FEV1 % months after 1st infusion (T6 L1) (n = 30) Results p (12,15) 0,92 (0,34) 35,93 37,67 (9,35) (10,69) Baseline (T0) (n = 29) Results (11,39) 0,220 0,010 months after 2nd infusion (T6 L2) (n = 29) Results p (12,58) 0,89 0,99 (0,29) (0,34) 35,55 40,48 (9,27) (10,77) 0,000 0,000 Table 0.8 Inflammatory markers before and after treatment Baseline (T0) (n = 30) Results Index IL- Rate of IL- months after 1st months after 2nd infusion infusion (T6 L1) (T6 L2) (n = 30) (n = 29) Results p Results P 16 23 (53,3) (76,7) Median 6,87 5,58 0,03 5,79 0,362 Median 2,24 2,55 0,045 2,52 0,02 IL-6/ Median 3,16 (1,23; IL-10 (Min; Max) 61,20) 0,007 (0,44; IL-8/ Median 6,39 (1,89; IL-10 (Min; Max) 487,89) Median 3,80 (1,12; (Min; Max) 6,84) (pg/ml) IL-6 (pg/ml) IL-10 (pg/ml) TNFIL-10 05 4.2 Characteristics of BM fluid and BM stem cells in COPD patients 4.2.1 Characteristics of BM fluid in COPD patients The average volume of BM fluid of the intervention group was 340.43 ± 43.43 ml, the lowest was 270 ml, the highest was 409 ml The results of analysis of the first 1ml of BM fluid showed that the number of nucleated cells reached 63.15 ± 43.69 (G/L) and the average number of MNCs was 20.62 ± 7.72 (G/L) L) Equivalent to the normal index of Vietnamese people published in 2001 the number of nucleated cells in the BM fluid is 57.36 ± 15.50 (G/L) When analyzing the cellular composition of the whole collected bone marrow fluid, the average number of nucleated cells was 21.47 ± 6.34 (G/L) and MNCs was 5.68 ± 2.07 (G/L), equivalent to previous studies In each study, research subjects have their own characteristics related to age, background disease, treatment goals for different diseases, in addition, there are no guidelines for cell treatment procedures As a standard for pathology, criteria for bone marrow aspiration and cell types in bone marrow fluid may vary between studies 4.2.2 Characteristics of autologous BM stem cells of patients with COPD after extraction The number of nucleated cells before extraction averaged 21.47 G/l, after separation increased to 59.75 G/l, nucleated cells were concentrated 2.5 times with p 21 < 0.0001 The survival rate of nucleated cells after separation was 97.43% (90-99%), this result achieved the goal of setting the survival rate of cells after separation > 75% The average number of nucleated cells in the stem cell bag after extraction was 4931.73 ± 1883.40 x106 The average number of MNCs after separation in our study was 1255.10 ± 521.68 x 106 After extraction, the percentage or concentration of CD34 and MSCs was quite high in the obtained stem cells, but there was a large difference between them The average CD34+ cell concentration was 383.67 ± 336.43 cells/µl, the least amount was 43.78 cells/µl, the maximum vesicle was 1701 cells/µl, the CD34+ cell survival rate was also significant strong fluctuations similar to the concentration with the lowest rate of 32.95%, the highest rate of 98.06% CD34+ imprinted cells are important progenitor cells that have a high capacity to multiply and differentiate and can be transformed into pluripotent stem cells when needed In fact, with each different clinical trial, on different patient subjects, for the treatment of different diseases, the procedure for collecting BM fluid and stem cell extraction as well as the characteristics of the obtained stem cell graft is also the same In our study, due to some objective and resource-related reasons, we were unable to perform cell culture, but only identified MSCs mesenchymal stem cells by the markers CD105, CD90, CD73, and no hematopoietic cell markers based on flow cytometry This is a cell that has the ability to stimulate tissue cells to proliferate so it heals damage The amount of MSCs in normal marrow is 0.001-0.01% and in umbilical cord blood 2-3 cells/108 mononuclear cells, in our study on average 0.04% fluctuating 0.0027 %- 0.398 % This rate is within good value The average survival rate of MSCs was 86.56% with vesicles reaching 100% In the treatment of COPD, stem cell therapy is an adjunct therapy, not a replacement therapy, so the amount of cells required for treatment is not as high as for stem cell transplant diseases CD34 and MSC cells are both located in the mononuclear cell layer, so the number and proportion of monocytes will determine the efficiency of stem cell graft separation 4.2.3 Characteristics of cryopreserved BM stem cells in COPD patients Cryopreservation has the potential to affect stem cell function, resulting in stem cell death Trypan blue staining immediately after thawing, the result obtained is the average survival rate of nucleated cells after thawing is 81.90 ± 5.99%, the lowest is 22 75% Cell survival is affected by many factors DMSO is a preservative solution, and cooling or thawing procedures can also be a factor After thawing, the cells are very sensitive, in addition, at high temperature, the toxicity of DMSO to the cells increases, so many studies have shown that the rate of cell death increases gradually over time The volume of stem cells after thawing was small, only 47.5ml, so all patients had an infusion time