Roosternourish msc

Human mesenchymal stromal cells (MSCs) were isolated from single donor bone marrow (Lonza, Walkersville, MD, USA) based upon their adherence to tissue-culture treated flasks in standard conditions. MSCs were cultured in minimal essential media-α (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2.5 ng/mL rec. human FGF-2 (Waisman Biomanufacturing, Madison, WI, USA), 10% v/v Hyclone FBS (Cytiva, Marlborough, MA, USA) and 1% v/v antibiotic- antimycotic (Thermo Fisher Scientific) at 37°C/5% CO₂.
Working Cell Bank (MSC-WCB, Passage: 2) and Cell Therapy Product (MSC-CTP, Passage: 3) stocks of MSCs were used for the entirety of this study. Once thawed, cells were seeded at a density 3,000–3,500 cells/cm² and weaned to xeno-free conditions using RoosterNourish-MSC (Rooster Bio, Fredrick, MD, USA). After 4 days, MSCs were dissociated from flasks using TrypLE Express Enzyme (Thermo Fisher Scientific) and counted using an NC-202 automated cell counter (ChemoMetec, Allerod, Denmark). Cell suspensions were centrifuged at 1100 rpm for 5 min and resuspended for encapsulation.

Prior to encapsulation, cell suspensions were mixed with VitroGel-MSC (TheWell Biosciences, North Brunswick, NJ, USA) to a total volume of 6 mL in a 1:2 v/v ratio according to the manufacturer’s recommendations. This hydrogel precursor solution was loaded into a 10 mL syringe and mounted vertically onto a syringe pump (Harvard Apparatus, Halliston, MA, USA). Microcapsule generation was performed using a VARV1 Encapsulation Unit (Nisco Engineering AG, Zurich, Switzerland) at a voltage supply of 4.55 kV and 20 mL/h syringe pump flow rate. For all encapsulations a 28G nozzle supplied by Nisco Engineering AG was used and placed at a constant height of 3.2 cm from the collection basin. Electrosprayed microcapsules were allowed to crosslink for 4 hours in the collection basin containing 80 mL of RoosterNourish-MSC (Rooster Bio). After 4 hours, microcapsules were transferred to a PBS0.1 vertical-wheel bioreactor (PBS Biotech, Camarillo, CA, USA) and increased to a final volume of 90 mL. Bioreactors were maintained at an agitation rate of 25 rpm and 37°C. On day 3, a xeno-free RoosterReplenish-MSC-XF (Rooster Bio) was added at 2% v/v and the agitation rate was increased to 30 rpm.