Tff membrane filter system

The SHED-CM was generated as follows: when passage 3 SHED cells reached 80% confluence in a 150 cm² cell culture flask, the medium was changed with 10 mL Dulbecco’s modified Eagle’s medium (DMEM) and 20 mL Hanks’ balanced salt solution (HBSS) in each flask, then incubated for 72 h at 37 °C in an atmosphere containing 5% CO₂ at 100% humidity. The medium was collected and centrifuged for 3 min at 2500 rpm. In order to collect proteins with molecular weights between 5 and 30 kDa, CM was collected in a sterilized beaker, and further concentrated by Tangential Flow Filtration (TFF) membrane filter system (Millipore) with a 5 kDa and 30 kDa cut-off unit (Millipore) alternatively for 3 h following the manufacturer’s instructions. The protein concentration in SHED-CM was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted to 100 ± 2.8 µg/mL with normal saline. Quantitation of each constituent was analyzed by ELISA array, Quantibody® Human Cytokine Antibody Array 4000 (RayBiotech), following the manufacturer’s instructions. This array can detect 200 target proteins, including human inflammatory factors, growth factors, chemokines, receptors, and cytokines.

Conditioned medium (CM) was generated as follows: 80% confluent passage 3–5 DPSCs in a 150 cm² cell culture flask (Corning) were fed with 10 mL DMEM/F12 and 10 mL PBS in each flask, then incubated for 48 h. The medium was collected and centrifuged for 3 min at 2500 rpm. In order to collect proteins with molecular weights between 5 and 30 kDa, CM was collected in a sterilized beaker and further concentrated by using a TFF membrane filter system (Millipore, Burlington, MA, USA) with a 5 kDa and 30 kDa cut-off unit (Millipore) alternatively for 3 h, following the manufacturer’s instructions. The protein concentration in DPSC-CM was measured using a BCA Protein Assay Kit (Pierce, Rock-ford, IL, USA) and adjusted to 27 ± 2.1 μg/mL with Normal saline. Quantitation of each constituent was done after TFF membrane filtration by using Qubit Fluorometric Quantification and Raybiotech ELISA Array.