aggregation (see Note 19) or stored at 4 C for up to a
week until analysis.
(e)
Following sedimentation of the MCs in the 15-mL cen-
trifuge tube, gently resuspend the MCs in 1 mL of
pre-warmed DPBS and transfer the suspension to a
2-mL microtube (Eppendorf®) using a 1-mL pipette and
a wide orifice (1.5 mm) pipette tip.
(f)
Allow the MCs to sediment, then remove and replace the
DPBS with 0.5 mL of pre-warmed TrypLE Select 1.
(g)
Place the 2 mL microtube horizontally in the incubator
with integrated orbital shaking platform at 130 rpm,
25 mm, 37 C for 7 min (see Note 7).
(h)
Resuspend the suspension 10 times using a 1-mL pipette
and standard pipette tip, then determine viable cell density
(see Note 2). Attachment efficiency may then be calcu-
lated by dividing the result by the cell density of the
inoculum. Be sure to account for the concentration factor
(approximately 10:1, see Note 13).
2. Standard samplings of 5 mL (see Fig. 4) are performed every
24 h post-inoculation for the duration of the cultivation.
Sample processing
Sample processing
Pellet
Pellet
Pellet
Supernatant
Aliquote 2
2 mL
Aliquote 1
1 mL
Aliquote 3
2 mL
Trypsination
Filtration
Trypsination
DAPI analysis for
determination of cell
attachment and distribution
Flow cytometry for
cell quality control
Cell density and
viability analysis
Substrate and
metabolite analysis
Pellet
Pellet
Pellet
Supernatant
Aliquote 2
2 mL
Aliquote 1
1 mL
Aliquote 3
2 mL
Trypsination
Filtration
Trypsination
DAPI analysis for
determination of cell
attachment and distribution
Flow cytometry for
cell quality control
Cell density and
viability analysis
Substrate and
metabolite analysis
Sample processing
Pellet
Pellet
Pellet
Supernatant
Aliquote 2
2 mL
Aliquote 1
1 mL
Aliquote 3
2 mL
Trypsination
Filtration
Trypsination
DAPI analysis for
determination of cell
attachment and distribution
Flow cytometry for
cell quality control
Cell density and
viability analysis
Substrate and
metabolite analysis
Fig. 4 A general overview of the sample work-up. The homogenous 5 mL sample is divided into three aliquots,
which are subsequently processed separately to determine MC aggregation, substrate, and metabolite
concentrations, as well as cell distribution, density, viability, and quality using various analytical approaches
such as staining with DAPI
Mesenchymal Stem Cell Expansion at Benchtop-Scale
101