Figure 3 shows the cell concentration profile versus time and cell nuclei stained with DAPI after 5, 10, and 20 days of culture
in stirred agitation spinner flasks. The cells readily propagated and after 3 days cells were found attached to nearly every
microcarrier. This was a very important result because it shows that the MSCs can jump from one microcarrier to another under
stirred agitation. Because only around 4% of microcariers that were seeded under static conditions were still in the culture
after 10 days, and yet the day 20 image shows more than 4% of the microcarriers populated with cells, the cells must have
traversed from a confluent surface to a fresh surface to populate the fresh microcarriers.
Figure 3: (A) Cell nuclei and (B) cell culture concentration profile after 5, 10, and 20 days of culture in stirred agitation
After it was shown that MSCs could propagate under stirred agitation, studies relating to the scale-up from the 125-mL spinner
flask to the Mobius CellReady 3L bioreactor were undertaken. MSCs on microcarriers that were still in the exponential growth
phase were transferred from spinner flasks (200 mL at 100,000 cells/mL) to the single-use Mobius CellReady 3L bioreactor which
contained 800 mL of media with fresh microcarriers that were at temperature and agitating at 25 RPM.
On day three, one liter of media with fresh beads was added to the one liter culture and the impeller rate was increased to
40 RPM to keep the microcarriers suspended. The cell concentration profile of cells on microcarriers growing in the Mobius
CellReady 3L and images of cell nuclei on microcarriers at various time points are depicted in Figure 4. Cell number increased
by 5.2-fold over the five day culture period.
Figure 4: (A) Increase in cell number in single-use bioreactor. (B) By the seventh day of culture, greater than 90% of the
microcarriers had mesenchymal stem cells growing on them.
This growth rate is similar to the 48 h doubling time exponential curve for the first five days; the cell concentration then
drops off this pace slightly by the seventh day. As shown in the images, after one day of growth in the Mobius CellReady 3L
bioreactor there are still fresh beads that have not been populated and microcarriers that are near confluence. The latter
are probably beads that were transferred from the spinner flask, but there are also several microcarriers that have nuclei,
indicating new growth. The day four image shows many microcarriers with many MSCs, but there are also some naked microcarriers,
most likely introduced when the number of microcarriers was diluted 1:1 with new micorcariers with the day three media addition.
In the day seven image, propagation of cells from bead to bead is most evident; a culture that begins with a minority of confluent
microcarriers (10%) has MSCs on greater than 90% of the microcarriers a week later. Some small aggregates of microcarriers
(2–5 beads) were observed, but because the static 3D petri dish cultures also contained small aggregates which was the model
for 3D growth, these small clumps were not discouraged. The cell concentration measured by counting nuclei lysed off the microcarriers
and the images of nuclei on the microcarriers give a strong indication that the cell number was increasing and that the cells
were propagating to fresh microcarriers. These analyses, however informative, do not supply information regarding the health
of the cells, nor do they indicate if these cells have the typical characteristics of MSCs. Accordingly, further characterization
studies were performed.
To better understand the health of the cells and to determine if the cells are active, several additional parameters were
measured. Daily supernatant samples from the single-use bioreactor were collected and analyzed and the profiles of nutrients
and metabolites in the media during the culture were generated (see Figure 5). Glucose levels decreased from 0.79 to 0.48
g/L between day one and three; after the media addition, levels fell again from 0.57 to 0.02 g/L between days four and seven.
The average specific glucose consumption rate over the culture was 2.7E-09 g/cell-day. Lactate levels increased steadily from
0.34 to 0.68 g/L between days one and three, and again after the media addition, from 0.51 to 1.0 g/L over the last three
days. The average specific generation rate of lactate was 1.9 E-09 g/cell-day during the culture. Because of the decreasing
glucose levels, it was decided to perform characterization, viable recovery, and differentiation studies after five days in
the Mobius CellReady 3L bioreactor. Cultures longer than five days may require more media additions or perhaps higher levels
of glucose in the growth media (low glucose was used for this study) to maintain suitable levels of glucose for exponential
cell growth. Showing that the MSCs are consuming glucose and producing lactate over the first five days of the culture indicates
that the culture is healthy, but it does not indicate how the cells might perform after being removed from the microcarriers
after the 3-L culture.
Figure 5: (A) Analysis of glucose and (B) lactate levels from the 3-L single-use bioreactor.