stock solution. The stock solution may be stored at 4 ^C for up

to 6 months if required. The stock solution must be further

diluted at a ratio of 1:40 using WFI to achieve the working

solution with a final Synthemax™II-SC concentration of

0.025 mg mL^1 prior to application. Add the working solution

to the cultivation vessel under sterile conditions and incubate

for 2 h at room temperature. For more information, consult

the self-coating protocol available on the manufacturer’s

website.

9. Enzymatic dissociation of the adherent hASCs can be achieved

by removing the cell culture medium supernatant from the

cultivation vessel, then washing with and subsequently remov-

ing pre-warmed DPBS. Finally, add 10 mL of TrypLE Select

1 (Gibco^®) to the T75-Flask, and incubate the cultivation

vessel at 37 ^C for 5 min. For further information regarding the

volumes of TrypLE Select 1 required for other cultivation

vessel types, please consult the online protocol supplied by the

manufacturer. Cell dissociation should be observed by micro-

scope directly following incubation. If this is not the case and a

large percentage of cells remain attached to the substrate,

incubate the cultivation vessel for an additional 5 min or gently

tap the side of the cultivation vessel using the back of a scissor

to facilitate the dissociation process.

10. ProNectin^® F MCs (Pall^® SoloHill^®) consist of a cross-linked

polystyrene core (diameter of 125–212 μm) and a coating

containing multiple copies of the human fibronectin RGD

attachment domain. See the publication by Rafiq et al. [30]

for more information. Recently, these MCs were transferred to

Sartorius AG as part of a divestiture agreement, after which the

product line was discontinued. A suitable commercially avail-

able coated polystyrene alternative with the same dimensions

and density as the ProNectin^® F MCs are the Low Concentra-

tion Synthemax^® II MCs (Corning^®).

11. The dissociated cells suspended in TrypLE Select 1 should be

transferred into a 50-mL centrifuge tube (Corning^®) contain-

ing 20 mL of pre-warmed cell culture media (see Note 6),

centrifuged at 300  g for 5 min, resuspended in 5 mL fresh

pre-warmed culture media (see Note 6) and their cell density

determined (see Note 2).

12. The volume of cell suspension required for inoculation follow-

ing each passaging step may be calculated using the viable cell

density determined using an appropriate cell counter (see Note

2). Simultaneously, additional information regarding cell qual-

ity may be ascertained using flow cytometry (see Note 4).

13. Gravimetrical measurements may be used to assist in ensuring

the concentration of the MC suspension either has remained

constant or has been sufficiently concentrated (or diluted)

106

Misha Teale et al.