Abstract 3
Introduction
DUOC‐01 are macrophage‐like cells derived from monocytes in cord blood. These cells are active in numerous preclinical models of multiple sclerosis (MS), reducing the progression of paralysis in experimental autoimmune encephalomyelitis and promoting remyelination after cuprizone. DUOC‐01 is currently being tested as an intrathecally dosed bridging therapy in children with demyelinating leukodystrophies undergoing unrelated donor umbilical cord blood transplantation after myeloablative conditioning. DUOC‐01 is a potentially attractive therapy for MS patients who experience destruction of myelin sheaths as part of their disease. To meet the dose requirement for adult patients, we set out to test if expanding myeloid progenitors could increase the yield of DUOC‐01.
Objective
We aimed to optimize the DUOC‐01 production pipeline to grow large numbers of DUOC‐01 per cord blood unit. We added a preliminary step, expanding myeloid progenitors using MethoCult H4435, and utilized these expanded cells to manufacture DUOC‐01.
Methods
Cryopreserved cord blood units were thawed in a 37°C water bath. Blood was extracted from the bag using a 16‐gauge‐needle and diluted to 1/8 of the initial concentration through serial 2× dilutions using a dextran solution. The diluted blood was spun at 4°C at 800 g for 30 minutes. The supernatant was aspirated, and the pellet was resuspended in PBS containing 1% HSA. After an additional spin at 300 g for 15 minutes, the cell pellet was resuspended, and the cells cultured in MethoCult H4435 for 8 days to select and expand progenitors. The expanded cells were then transferred to T75 flasks to grow DUOC‐01.
Results
The expansion with MethoCult resulted in more progenitors. At small scale, the DUOC‐01 yield increased up to 29‐fold compared with cells grown without the expansion. Surface markers of DUOC‐01 produced with or without the expansion were comparable as measured by flow cytometry.
Discussion
We describe a novel approach to manufacturing of DUOC. The expansion of progenitor cells prior to DUOC culture yields much higher numbers of DUOC‐01 at the end of manufacturing. This may enable the use of higher doses or multiple doses of DUOC to treat an individual patient and should increase the dosing potential of DUOC in patients with MS.