09.2018 | THE SCIENTIST 61LI ET AL.,
BIOFABRICATION
, 10:025006, 2018, DOI:10.1088/1758-5090/AA6B5, CC
BY
3.0
are what many scientists would like to
imitate with iPSCs. They lack epigene-
tic marks that program cell fates, mak-
ing them fully pluripotent. In contrast,
most iPSC cultures are primed stem
cells. They represent a more mature
embryonic tissue, the epiblast that forms
the external layer of an embryo. Primed
iPSCs often contain epigenetic marks,
though the genes turned on and off are
fairly random, says Bamdad.
That means not all cells in an iPSC
culture are truly pluripotent; many are
already on their way to one cell fate or
another. “It is difficult to make primed
stem cells differentiate into mature, func-
tional cell types that we need for therapeu-
tics,” says Bamdad.
The reason iPSCs tend to turn out
primed instead of naïve, she adds, is that
most protocols use media containing fibro-
blast growth factor (FGF). “FGF, which is
used to grow these cells during reprogram-
ming and after, prevents them from going
all the way back to the earliest state that we
call naïve.” Only about 15 percent of iPSCs
generated with FGF turn out to be “bona
fide” iPSCs, says Bamdad.
Minerva’s AlphaSTEM cell culture
products provide a more native environ-
ment, she says, because they use a dif-
ferent growth factor, NME7 (Stem Cells,
34:847–59, 2016). NME7 is typically
expressed in very early embryos, so it
better mimics the naïve stem cell niche.
Using the recombinant NME7AB, Bam-
dad says, yields better iPSCs. “By ‘bet-
ter,’ we mean works every time... bet-
ter functional profile, higher yields,” she
says. “They all can become any cell type.”
With AlphaSTEM Naïve HPSC Medium($295/500 mL), researchers can repro-
gram the majority of cells in their cul-
tures to a pluripotent state, she adds.
Other pro-naïve solutions exist, such
as 2i and 5i media formulations. These
rely on leukemia inhibitory factor (LIF)
from mice and other biochemical inhib-
itors. While these media do yield cells
that are more naïve than primed cells,
Bamdad notes they can create instability
in chromosome numbers, and she’s not
sure how well they mimic true embry-
onic stem cells.
Minerva researchers say naïve iPS cells
could streamline cell production, improve
gene-editing efficiency, and boost the over-
all quality of therapeutic stem cells. “If you
start with better cells, everything’s going to
be easier,” Bamdad says.ADD DIMENSIONS
Another way to improve the stem cell cul-
ture environment is to consider its topo-
graphy. Cells in the lab often grow in
monolayers on flat glass or plastic. All
those plates require a lot of space, media,
and time to care for them; it’s highly inef-
ficient. One solution is to grow cells in sus-
pension, in large flasks or bioreactors. (See
“Easier Ways to Grow Stem Cells,” Septem-
ber 2017, The Scientist.)
But big bioreactors create problems, too,
says Lei. At first, the stem cells begin to clus-
ter together into little spheroids. That’s good;
stem cells don’t typically grow alone in situ.
But in bioreactors, those spheroids eventu-
ally grow and coalesce into big aggregates.
Nutrients and oxygen can’t reach the cells in
the center of the aggregate, and wastes don’t
easily diffuse out of the blob. “That makes
the cell culture very inhomogeneous,” saysLei. Aggregation can slow growth, induce
apoptosis, or alter differentiation patterns.
Stirring the tank minimizes aggre-
gation, but such agitation creates a shear
force that can kill cells, especially sensi-
tive human stem cells. Lei’s lab has found
that up to 40 percent of stem cells grown in
spinning suspension die every d ay.
Lei sought a way to manage stem cell
cluster size while protecting the frag-
ile cells from shear forces. He developed
a hydrogel tube in which stem cells can
grow (Biofabrication, 10:025006, 2018).
The lab uses a custom micro-extruder to
make the tubes, which are up to 400 μm
in diameter. As they extrude the alginate
for the tube through a cylindrical channel
(shell flow), they simultaneously pump a
cell suspension through its center (core
flow). These materials land in a calcium
chloride buffer, which makes the alginate
solidify into tubes around a cell suspension
core. With the AlgTubes, his cultures look
like vats of udon noodle soup, Lei says.
The tubes constrain the size of the cell
aggregates so that nutrients and oxygen can
still diffuse in, and wastes can filter out. The
hydrogel barrier insulates cells from shear
forces in moving media.
Lei’s results indicate stem cells are much
more comfortable in the tubes. Fewer thanTOTALLY TUBULAR: Growing human embryonic
stem cells in hydrogel tubes protects them
from moving media and promotes health
and higher yields.If you start with better cells,
everything’s going to be easier.
—Cynthia Bamdad, Minerva Biotechnologies