NTCELL is comprised of choroid plexus cells encapsulated in alginate microcapsules. The encapsulation procedure produced alginate microcapsules (≈400 to 550 μm in diameter) that contained 1 to 4 clusters of choroid plexus cells, typically in a spherical, ovoid, or branched shape.
Choroid plexus was isolated from neonatal pigs (either sex, 7–10-days-old and 3.5–5.5 kg). The brain was dissected through the midline and the choroid plexus (CP) was removed and placed in HBSS (0-4°C), supplemented with 2% human serum albumin and processed as follows: (1) the tissue was minced, allowed to settle, and the supernatant was removed; (2) collagenase (1.5 mg/mL, in 5 mL HBSS at 0-4°C) was added and the tissues were allowed to sediment before removing the supernatant; (3) fresh collagenase (1.5 mg/mL, in 15 mL HBSS at 0-4°C) was added, warmed to 37°C, and stirred for 15-20 minutes; (4) the digested material was mixed with an equal volume of RPMI supplemented with 10% neonatal porcine serum, triturated gently, and passed through a 200-m stainless steel filter; (5) the suspension was centrifuged (500 rpm, 4°C for 5 minutes), the supernatant was removed, and the resulting cell clusters (50 to 200 µm in diameter) were gently resuspended in RPMI supplemented with 10% neonatal porcine serum; and (6) blood cells were removed by sedimentation for 25 minutes at 0-4°C before the supernatant was aspirated. The final preparation was adjusted to 3000 epithelioid clusters/mL in RPMI with 10% neonatal porcine serum and placed in nonadherent Petri dishes. Half of the medium was removed and replaced with fresh medium after 24 hours and again after 48 hours. Before encapsulation, the cell clusters were washed by sedimenting 3 times in 2% (0.75 mol/L) human serum albumin in HBSS at room temperature. The cells were encapsulated in alginate. Encapsulated cells were maintained in culture for 7 days before transplantation.
Encapsulation: Following the final sedimentation and removal of the supernatant, alginate (1.7% w/v, isotonic, 25°C) was added to the cell preparation to allow an encapsulation density of 50,000 cell clusters/ml alginate. Using a concentric nozzle system with an inner diameter of 575 μm and shear force generated by nitrogen flowing at 3 l/min, droplets were generated and allowed to settle into a 200 ml bath of calcium chloride (110 mM) which was stirred transiently to suspend the precipitated spheres. After a 5-min exposure, the supernatant was removed by vacuum aspiration, and the capsules were washed once with saline. The following coating steps were all carried out at a ratio of 2:1 (coating solution:capsule volume) under mild rocking agitation. Capsules were coated with a 0.1% (w/v) solution of polyornithine (PLO) for 10 min, followed by a wash and a secondary PLO coat (0.05% w/v, 5 min). After another wash, an outer layer of alginate was applied by exposing the surface to 0.17% (w/v) alginate for 6 min. Following a wash, the capsule core was dissolved with 55 mM sodium citrate (tribasic sodium citrate), and the capsules were washed one more time.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
