62 THE SCIENTIST | the-scientist.com
LAB TOOLSCAROTIET AL.,SCIREP,7:13334, 2017.10 percent die every d ay. Yields are higher,
with up to 500 million stem cells growing
in every milliliter of media within the tubes,
compared to 2 million to 5 million per mil-
liliter in suspension culture. Lei expects it
will be possible to scale that up. For exam-
ple, one could make 10 billion cells with just
two milliliters of space contained in tubes.
The cells in AlgTubes can be cultured for
more than 50 days, can expand 1,000-fold
in just 10 days, and can be differentiated
into a variety of cell types.
AlgTubes can last for months, and
when the researchers want to get the cells
back out, it’s easy. They use EDTA to che-
late the calcium ions in the hydrogel, and
the tubes fall apart.
Lei can provide scientists with detailed
instructions for making their own AlgTube
extruder, or his group can produce them
for collaborators. He’s started a company,
CellGro Technologies, to commercialize
the extruder.SYNERGIZE
If one method improves stem cell yields
and qua lit y, wouldn’t two be better?
That’s what Lyle tested with the mouse
MSCs she isolated from bone ma r row.A good protocol to grow and main-
tain mouse MSCs would be a boon for
research, she says, because scientists
already have so many genetically engi-
neered mouse lines. Researchers could
isolate MSCs from those lines, and
then determine how different genes are
involved in differentiation, she says.
Lyle read that basic fibroblast growth
factor (bFGF) worked well for MSCs, likely
due to its pro-growth and pro-survival sig-
naling. (FGF wouldn’t be a bad choice in
Lyle’s case, because she wasn’t trying to
grow naïve cells.) She’d also seen reports
that hypoxia would help. That’s probably
because oxygen levels in the bone marrow,
where MSCs naturally exist, are around 5
percent, much less than the atmospheric 21
percent that most stem cells are cultured in.
The research team tested four treat-
ments: normoxia and hypoxia, each with
or without bFGF. Then, when the cells
covered much of the dish surface and were
ready for their first passage, the scientists
measured the concentration of cells per
square centimeter. With atmospheric oxy-
gen and no bFGF, the cells were ready to
passage in 39 days, and there were about
6,600 cells per square centimeter. WithbFGF, the cells achieved the required con-
fluence within 27 days, and they crowded
together at 43,000 per square centimeter.
With hypoxia and no bFGF, the cells were
ready after 20 days, and their numbers rose
to 99,000 per square centimeter. But with
both hypoxia and bFGF, the cells were con-
fluent at 14 days, and the concentration was
the highest—220,000 per square centime-
ter (Sci Rep, 7:13334, 2017).
Overall, the MSCs in low-oxygen,
bFGF conditions grew 2.8 times faster
than under standard protocols, and
reached passage 3, when cells are typi-
cally ready to test, within one month
instead of the usual two or three. They
were less likely to senesce, and retained
their ability to multiply or differentiate
until at least passage 11.
The results of the dual treatment are
“crazy good,” says Lyle, “I think because it’s
closer to what their native niche is.”
Lyle has already used her protocol to
generate MSCs from transgenic mice that
make luciferase or GFP. Scientists could
then engineer these MSCs and implant
them into mice without the luminescent
or fluorescent markers, and track where
the glowing cells end up and how long
they survive.
And she’d be happy to add other treat-
ments to improve yields and efficiency. “It
doesn’t have to be a one-pronged approach,”
says Lyle. “It can be multifactorial.”
Indeed, Lyle adds, current stem cell cul-
ture protocols probably haven’t reached any-
where near the optimal conditions for effi-
cient growth and division. “There is still a lot
of area for improvement,” she says. gTWO ARE BETTER THAN ONE: While both low
oxygen (right column) and basic FGF (bottom row)
improve growth of mouse mesenchymal stem cells,
the combination of the two (bottom right) works
the best.It doesn’t have to be a one-
pronged approach. It can be
multifactorial.
—Alicia Lyle, Emory University