A Modular Bioinstructive Platform For Additive-Free Control of Stem Cell Differentiation and Patterning
A Modular Bioinstructive Platform For Additive-Free Control of Stem Cell Differentiation and Patterning
This experiment has 3 factors with total of 6 conditions. Two bead types: smooth and dimpled. Two time points: day 3 and day 14. Two growth conditions for dimpled: control and KAAD. Fabrication of smooth and dimpled microparticles Poly(D,L-lactic acid) (PLA) microparticles (Ashland Viatel DL 09 E, Mn 56.5 kDa, Mw 111 kDa, IV 0.8–1.0 dL/g) were prepared using a solvent evaporation oil-in-water emulsion technique [PMID: 33096376, PMID: 37837904]. For fabricating smooth microparticles, a 20% (w/v) solution of PLA in dichloromethane (DCM; ≥99.8%, Thermo Fisher Scientific, USA) was homogenised (Silverson Machines Ltd., UK) at 3800 rpm for 5 min. The homogenised organic phase was then emulsified into 100 mL of an aqueous continuous phase containing 1% (w/v) poly(vinyl acetate-co-alcohol) (PVA; MW 13–23 kDa, #348406, Sigma-Aldrich). The resulting emulsion was stirred at room temperature to facilitate solvent evaporation. Microparticles were collected by centrifugation and washed with deionised water to remove residual PVA. After washing, microparticles were sieved using strainers (40–70 µm) (Greiner bio-one) and then freeze-dried for storage. For the fabrication of dimpled microparticles, the addition of fusidic acid (FA; 98%, #5552333, Thermo Fisher Scientific, USA) into the organic phase was used to create the topographical patterns. A 30% (w/v) ratio of FA to PLA was used, resulting in a total FA/PLA concentration of 10% (w/v) in DCM. FA-loaded microparticles were incubated in phosphate-buffered saline (PBS, Gibco) at 37 °C for 7 days for FA release, as previously detailed [PMID: 33096376].  Primary human mesenchymal stromal cell culture Primary human bone marrow-derived mesenchymal stromal cells obtained from 3 independent donors representing diverse demographic backgrounds were used. Three donor lots (designated as donors 1, 2 and 3) were obtained from RoosterBio (RoosterVial™-hBM-1M, MSC-003, RoosterBio Inc., USA). Cells were cultured in Dulbecco's modified Eagle's medium (#21969-035, Gibco) supplemented with 1% (w/v) l-glutamine (Gibco), 1% (w/v) penicillin-streptomycin (Gibco) and either 10% (v/v) fetal bovine serum (FBS; Gibco) for routine passaging or 2% FBS (referred to as serum-reduced medium). Each donor batch was maintained as an independent stock and cells were used between passages three and six. Tri-lineage differentiation potential hMSCs was confirmed using StemPro™ differentiation kits (Gibco, UK). Microparticles preparation for cell seeding Microparticles were placed in CELLSTAR® cell-repellent surface 96-well plates (Greiner Bio-One) and sterilised with UV light at 254 nm for 30 min at 4 × 104 mJ. Mass of smooth and dimpled microparticles were calculated to achieve a consistent surface area for cell attachment. Following sterilisation, the microparticles were conditioned in serum-reduced medium (2% FBS) for 1 h. Cells were seeded onto microparticles at a density of 1 × 104 cells/cm² and placed on a plate shaker for 15 min to ensure even distribution. For 2D controls, cells were seeded in tissue culture-treated 96-well plates (CytoOne®, Starlab). Cell viability and proliferation Cell viability was assessed three days post-seeding using the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (30002-T, Biotium, UK) according to the manufacturer's instructions. Briefly, 1 μM calcein-acetoxymethyl (calcein-AM) and 4 μM ethidium homodimer III (EthD-III) were added to each well. Imaging was performed using a ZEISS Cell discoverer 7 imaging system (ZEISS, Germany). Proliferation was assessed by measuring DNA concentration from cell lysates using the Quant-iT™ PicoGreen® dsDNA Assay Kit (P7589, Invitrogen, USA), following manufacturer's instructions. Cells were lysed with CelLytic™ M lysis buffer (C2978, Sigma-Aldrich) with two additional freeze-thaw cycles. Fluorescence was measured at λexc/λem 480/520 nm using a Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, USA). DNA content was measured by comparing to a standard curve generated from the supplied standards. hMSCs (N= 3) for RNAseq were cultured on smooth and dimpled microparticles, for 3 and 14 days. For KAAD-cyclopamine treatment, hMSCs were cultured in serum-reduced medium for 24 h, then treated with 300 nM KAAD-cyclopamine (ab142146, Abcam), an SMO antagonist.  Corresponding 2D and 3D vehicle-only controls, were prepared as 0.06% (v/v) DMSO in serum-reduced medium, shown to maintain the surface topographical features intact [PMID: 37837904], with the treatment media refreshed every 2 days. Total RNA samples were extracted using the RNAqueous™-Micro Kit (AM1931, Thermo Fisher Scientific) with minor modifications to prevent dissolution of microparticles in ethanol. Total RNA quality was assessed using the Agilent 4200 TapeStation system with RNA ScreenTape assays (G2991BA, Agilent Technologies Inc), and the RNA Integrity Number (RIN) was determined using TapeStation Analysis Software (v5.1; Agilent Technologies). Illumina stranded mRNA library