Cultispher s

Initial microcarrier screening was performed in 6-well plates to investigate eCB-MSC attachment to five different microcarriers: Cytodex 1 (GE Healthcare Cat# 17–0448-01), Cytodex 3 (GE Healthcare Cat# 17–0485-01), Cultispher S (Sigma Cat# M9043), Enhanced Attachment (Corning Cat# 3779) and Synthemax II (Corning Cat# 3781). The cells and microcarriers were inoculated into the wells at 6700 cells/cm² (microcarrier surface area) with 3.0 mL of 30%FBS-0bFGF medium. The 6-well plates were placed on a shaking platform (Scientific Excella e5) at 60 rpm with a ¾” diameter shaking orbit and cell attachment counts were performed at 1, 2, 3, 4 and 24 h.

Cytodex 1 (GE Healthcare), Cytodex 3 (GE Healthcare), SphereCol (Advanced BioMatrix), and Cultispher-S (Sigma) microcarriers were prepared in accordance with the manufacturer’s instructions. Cytodex 1, Cytodex 3, and Cultispher-S were sterilized by autoclaving at 121 °C for 20 min while SphereCol was supplied in sterile form. All microcarriers were washed three times in MSC growth medium before use. Characteristics of the distinct microcarriers used are summarized in Table 1.Table 1

Characteristics of microcarriers tested and their respective seeding conditions per heMSC-microcarrier construct at day 0 of differentiation

	Cytodex 1	Cytodex 3	SphereCol	Cultispher-S
Microcarrier characteristics
Diameter (μm)	147–248	141–211	100–400	130–380
Matrix	Dextran	Dextran	Type I Collagen	Gelatin
Charges/coating	Positively charged	Denatured collagen	–	–
Porosity	Microporous	Microporous	Microporous	Macroporous
Biodegradability	No	No	Yes	Yes
Surface area (cm2/mg dry weight)	4.40	2.70	–	15.0
heMSC-microcarrier construct-seeding conditions (spinner culture, data per construct)
Cell confluency	70%	70%	–	–
Total cell number	10.1 × 103	10.1 × 103	–	–
Microcarrier number	300	300	–	–
heMSC-microcarrier construct-seeding conditions (shake flask culture, data per construct)
Cell confluency	70%	70%	70%	70%
Total cell number	10.1 × 103	8.88 × 103	8.88 × 103	30.8 × 103
Microcarrier number	300	300	300	50

heMSC human early mesenchymal stromal cell

Microtissues were fabricated as described in our previous report [21] (link). CultiSpher S (Sigma) are macroporous porcine gelatin microcarriers with a diameter of 130∼380 mm and pore size of 10 mm. Microcarriers were rehydrated in Ca²⁺ and Mg²⁺ free PBS, autoclaved and rinsed once with PBS and twice with growth medium. Microcarriers were loaded at 2 mg/mL in 250-mL siliconized spinner flasks (Corning) in growth medium supplemented with 0.05 mg/ml of ascorbic acid-2-phosphate (Sigma) and 5×10⁻⁵ M of mercaptoethanol (Sigma). Cells were seeded at 5×10⁴ cells/mL using an intermittent stirring regime (3 min agitation at 30 rpm followed by 30 min settling, which was repeated for a total of 8 h). Culture was agitated at 50 rpm and maintained in growth medium for 8 days before medium was replaced with osteogenic medium consisting of DMEM supplemented with 10% FBS, 100 nM dexamethasone (Sigma), 10 mM sodium-β-glycerophosphate (Sigma), 0.1 mg/mL ascorbic acid-2-phosphate and 5×10⁻⁵ M of mercaptoethanol. Culture lasted for another 20 days and medium change was performed every 2 days.