Cytopore1

The Young’s Modulus of Cytopore1 (GE Healthcare, 17-0911-01), Cytodex3 (GE Healthcare, 17-0485-01), and two types of Microcarriers (CytoNiche, Beijing, Microcarriers-W01, Microniche)^{25 (link)–28 (link)} were measured by AFM and compared with the elasticity of natural bone marrow (Using Yong Modulus in the range of 0.3–24.7 Kpa)^{42 (link)–44 (link)}. The microcarriers were dispersed with Cell-Tak (Corning) and attached to a coverslip, while samples attached to the coverslip were mounted on an inverted fluorescence microscope (Zeiss Observer A1 stand) and measured using an AFM module CellHesion® 200 (JPK Instruments). The silicon tipless cantilever (ARROW-TL1-50, NANOWORLD) that was used to indent the samples had a nominal spring constant of 0.03 N/m and was attached with a silicon microsphere (20 µm in diameter) on the cutting edge. For each sample, AFM indentations at different locations (n ≥ 30) of the cell were performed with ~0.6 nN force at a 10 µm/s displacement rate. The effective Young’s modulus was obtained by fitting the force-displacement curve to the Hertz/Sneddon model.

The microcarriers we used were cytodex 1, cytodex 3, and cytopore 1 (GE, Boston, MA, USA). The microcarrier was washed for three times with D-Hanks and stored in DMEM/F12 with 10% FBS. To generate microcarrier-based culture, an adequate amount of microcarrier was added into a non-adherent culture plate to cover the bottom of the plate. ADSCs were trypsinized and then added on to the microcarrier. This culture was established after incubation for 2 h to facilitate the cell attachment to the microcarrier with several times of mixing. To monitor the cell proliferation on the microcarriers, ADSC-EGFP cells were cultured on three types of microcarriers, and the fluorescent signals were measured by the fluorometer (SpectraMax Gemini XPS, Molecular Devices, San Jose, CA, USA). The empty microcarriers were used as background controls.