Engineering bone-forming callus-organoid implants in a xenogeneic free differentiation medium

The field of tissue engineering aspires to provide clinically relevant solutions for patients through the integration of developmental engineering principles with a bottom up manufacturing approach. However, manufacturing of cell based advanced therapy medicinal products is hampered by protocol complexity, lack of non-invasive critical quality controls, and dependency on animal-derived components for tissue differentiation. We investigate a serum-free, chemically defined, xeno- and lipid-free chondrogenic differentiation medium to generate bone-forming callus organoids. Our results show an increase in microtissue homogeneity during prolonged differentiation and a high quality of in vivo bone forming organoids. The low protein content of the culture media potentially allows for monitoring of relevant secreted biomarkers as (critical) quality attributes . Together, we envisage that this xeno- and lipid-free chondrogenic medium is compatible with industrial scale-up and automation, while facilitating the implementation of non-invasive imaging and use of quality control parameters based on secreted biomarkers. Human periosteum-derived cells (hPDCs) were isolated from periosteal biopsies of 6 different donors of which one cell pool was created (ages of 20-32). The hPDCs were expanded until passage 7 at 37 °C, 5 % CO2 and 95 % humidity in Dulbecco’s modified Eagle medium (DMEM, Gibco, UK) with 10 % fetal bovine serum (South Afrika FBS, BioWest, France) and 1 % antibioticantimycotic solution (Invitrogen, USA). Medium was changed every 3-4 days. At a confluence of 80 %, cells were harvested with TrypLE Express (Life Technologies, UK). A microwell platform (AggreWell™800, STEMCELL Technologies Inc, Canada) was coated with Anti-Adherence Rinsing Solution (STEMCELL Technologies Inc, Canada) to avoid cell attachment. Homogenous microwell coating was ensured by centrifugation, followed by a washing step with basal medium before cell seeding. hPDCs were harvested with TrypLE Express (Life Technologies, UK) and seeded at 300 000 cells per well to contain 1000 cells per microwell upon sedimentation. The cells selfaggregated and were differentiated for 21 days. Two serum-free, chemically defined chondrogenic media were compared. Both contain LG-DMEM (Gibco) supplemented with 1% antibiotic– antimycotic (Invitrogen), 1 × 10-3 M scorbate-2 phosphate, 1 × 10-7 M dexamethasone, 40 µg mL-1 L-proline, 20 × 10-7 M of Rho- kinase inhibitor Y27632 (Axon Medchem), 100 ng mL-1 BMP2 (INDUCTOS), 100 ng mL-1 GDF5 (PeproTech), 10 ng mL-1 TGF-β1 (PeproTech), 1 ng mL-1 BMP-6 (PeproTech), and 0.2 ng mL-1 basic FGF-2 (R&D systems). Xenofree chondrogenic medium (xfCM) was then supplemented with ITS Premix Universal Culture Supplement (containing 6.25 µg mL-1 insulin, 6.25 µg mL-1 transferrin and 6.25 ng mL-1 selenious acid, Corning), while chondrogenic medium (CM) was supplemented with ITS+ Premix Universal Culture Supplement (containing 6.25 µg mL-1 insulin, 6.25 µg mL-1 transferrin and 6.25 ng mL-1 selenious acid, 5.35 µg mL-1 linoleic acid and 1250 µg mL-1 bovine serum albumin to solubilize the linoleic acid, Corning). Half of the medium was changed on days 3, 7, 10, 14, and 17.