2.1. Patient Enrolment
A 43 years old male patient suffered from chest pain and was short of breath for two weeks and was admitted into acute care unit. The patient was diagnosed with acute myocardial infarction and subjected to angioplasty and stent implantation followed by MSC administration. A written conformed consent was obtained from the patient. The protocol was approved by the Institutional Board of Medical Ethics at the hospital.
MSCs were isolated from mononuclear cell population in the bone marrow. The isolation method of mononuclear cells was previously described [11,12]. Briefly, under local anesthesia, bone marrow (10 ml) was aspirated from the posterior iliac crest using a heparinized syringe. Meanwhile, 30 ml of venous blood was taken for serum extraction. Bone marrow suspension was loaded on the top of an equal volume of Ficoll (density 1.077 g/ml, Sigma) preloaded centrifuge tube and centrifuged at 1800 rpm for 20 min. The top layer of mononuclear cells was collected and washed with Dulbcco modified Eagle's medium (DMEM, Gibco) three times. The isolated cells were suspended in DMEM/15% fetal bovine serum (FBS, Hang Zhou Biology Technology Company) supplemented with hPDGF (10ng/ml, Sigma) and hEGF (10ng/ml, Sigma), and then, seeded into 50 cm2 flasks at a density of 106 cells/cm2. The cells were cultured in a 95% humidified incubator at 37oC with 5% CO2 in air. After 5 days, non-adherent cells were removed by replacing the medium. Attached cells developed into colonies within 3-7days. When these primary cultures of MSCs had reached 80-90% confluence, the cells were harvested using 0.25% trypsin (Gibco) and subcultured at a ratio of 1:3 for three passages. On the third passage, FBS and growth factors were removed from culture medium and replaced by 3% inactivated autologous serum.
2.3. Viability Assay, Microbiological Test and Cytogenetic Analysis
The cell viability was determined by trypan blue exclusion. Before transplantation, a sample of MSC suspension (10^l containing ~104 cells) was incubated with 5% Trypan blue at room temperature for 5 min. The percentage of non-stained cell number/total cell number was taken as the cell viability. Viability of >95% was one of the required criteria for the cell to be released for transplantation.
Gram staining and bacteria culture were taken as microbiologic tests. Negative Gram bacteria smear test and negative bacteria culture were required for MSC application.
After three passages of in vitro expansion, MSC cytogenetic analysis was performed by conventional staining and G-banding techniques as described in our previous report .
To identify the stem cell population, harvested MSCs were stained with saturating amounts of monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC): CD34-FITC (negative marker, Invitrogen) and CD44-FITC (positive marker, Invitrogen). The staining procedure was described in the manufacture's instruction. At least 20,000 events were analyzed by flow cytometry, E6155 (BD FACS Calibur).
MSCs were administrated by direct intrcoronary infusion and intravenous infusion sequentially. Heparinization and filtration (40^m cell strainer, BD Falcon) were carried out to prevent cell clotting and microembolization during intracoronary and intravenous transplantation. For the primary administration, 6.08 x 10' MSCs in 20ml were given just after stent implantation via the same angioplasty balloon catheter. All cells were directly placed within the infarct-related artery. Two weeks later, the autologous transplantation was boosted by intravenous infusion of 3.42 x 108 MSCs.
2.6. Assessment of Myocardial Perfusion and Cardiac Function
The heart function was determined using 2 D Doppler Echocardiography (IE33, Philip). Myocardial perfusion was assessed using 99mTc-tetrofosmin single-photon emission computed tomography (SPECT, DS7, Sophy). All examinations were taken before and after MSC administration. The patient was also regularly followed up for six months.
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