Sanne Hoeks

Chapter 4 54 METHODS Sample collection Between June 2011 and January 2013, 7 children were enrolled in the study. Infant characteristics are presented in Table 1. All mothers were healthy and had uncomplicated pregnancies. All children were prenatally diagnosed with a different severity of orofacial cleft that required 1 or multiple chirurgical corrections. Blood samples were drawn from the umbilical cord directly after birth and from peripheral blood during surgical interventions that took place at regular intervals after birth. Additional control samples were taken by venipuncture from 6 unrelated healthy volunteers. This study was approved by the local medical committee of the University Medical Center Utrecht (08-331). Written informed consent was obtained from all parents before inclusion in the study. All procedures adhered to the principles of the Declaration of Helsinki. Infant characteristics Mode of delivery - Vaginal (%) 71 - Cesarean section (%) 29 Sex - Female (%) 29 - Male (%) 71 Prematurity (%, GA below 37 wk) 14 (GA 34 +4 wk) Birth weight (g), mean ± SD 3307 ±819 Infant samples CB (%) 86 3 mo (%) 57 6 mo (%) 43 12 mo (%) 43 TABLE E1. Infant and sample characteristics. GA, gestational age. Cells and culture conditions After collection of the blood samples, CBMCs or PBMCs were isolated within 24 hours by using Ficoll-Isopaque density gradient centrifugation (Ficoll-Isopaque, Pharmacia, Upsalla, Sweden). Cells were washed and frozen in 90% FCS and 10% dimethyl sulfoxide at −80°C. Cells stored for more than 1 month were transferred to −150°C. All samples from one donor were thawed simultaneously together with a sample from an adult donor. Cells were subsequently aliquoted for different experiments.