Third-party MSC preparation was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Following good manufacturing practice (GMP), the cell preparation and culture processes were performed under standardized and aseptic conditions at the Stem Cell Laboratory Facility of the Biotherapy Center at our hospital [12]. Fresh UCs were obtained from healthy pregnant women and immersed in 4 °C phosphate-buffered saline (PBS). The umbilical cords were washed twice with PBS to remove the remnant blood until they became white, cut into 10 mm3/piece in 0.1% type I collagenase with CaCl2 (3 mM) containing 0.1% hyaluronidase (Invitrogen, USA), and then incubated on a shaker (220 rpm) at 37 °C for 4 h digestion. Subsequently, the isolated cells were cultured in low-sugar Dulbecco’s modified Eagle’s medium (1 g/L DMEM, Gibco, Life, Austria) with 10% fetal bovine serum (FBS, Gibco, Life, Austria) in a humidified atmosphere of 5% CO2 and 37 °C. The medium was refreshed every 3 days to remove nonadherent cells. To assess the phenotype on the cell surface, flow cytometric analysis of CD105, CD73, CD44, CD90, CD45, CD34, CD166, and CD29 was performed. The differential potential was detected according to criteria established by the 2006 International Society of Cellular Therapy, which investigated their ability to differentiate into osteocytes and adipocytes [15]. At 70–80% confluence, MSCs were passaged by trypsin treatment. MSCs were collected and clinically used at passages 3–5. Before injection, the cells were tested again and confirmed to be negative for HBV, HCV, HIV, syphilis, mycoplasma, fungi, and endotoxins.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.