Page 354 - IJB-9-2

International Journal of Bioprinting Laser bioprinting of hiPSC-derived neural stem cells and neurons

or accelerates neuronal network formation. Our results that have been cultured under neuronal differentiation
show a clear effect of pre-differentiation on cell viability and conditions for 5, 10, or 20 days before printing as neurons or
behavior after printing and contrary to our expectations, as d10 neurons after 10 days of differentiation.
a stronger effect of added astrocytes on un-differentiated After most printing experiments, cells were cultured
NSCs than on pre-differentiated ones. under neuronal differentiation conditions. While neurons
1.1. Notation were directly further kept under neuronal differentiation
All cells used for this study were NSCs derived from conditions, printed NSCs were kept under normal NSC
hiPSCs. Some of these NSCs were differentiated to neurons culture conditions for 2 days after printing before the
or astrocytes. This produced mixtures of cells from different differentiation was started to enable distinction between
differentiation stages. the effects of medium change and printing. This results in
the need for a distinction between differentiation before
To avoid repetitive long descriptions for these mixtures, and after printing, for which we will use the terms “pre-
we use the following notation for neuronal differentiation differentiation” and “post-differentiation,” and a distinction
of NSCs: For cells printed as NSCs and differentiated between the time since printing and the time duration of
after printing, we use the term “post-differentiated NSCs” differentiation after printing, which is the same for neurons
to distinguish them from “NSCs” that were already (pre-differentiated NSCs) but differs by two days for NSCs
differentiated before printing (pre-differentiated), which (non-pre-differentiated NSCs).
we call “neurons” or, e.g., “d10 neurons” for 10 days of
pre-differentiation. Additionally, we use the notation “(day Therefore, we will use the following notations for
A pp, diff B/C),” which reads “cells assessed A days post- neuronal differentiation of NSCs: For not pre-differentiated
printing with B days of pre-differentiation and C days of cells we will use the term ‘”NSCs” and “post-differentiated
post-differentiation.” For a more detailed description and NSCs,” when differentiated after printing. For astrocytic
rationale, see section 2.2. pre-differentiated NSCs, we use the term “astrocytes,” and
for neuronal pre-differentiated NSCs, we use the term
2. Materials and methods “neurons,” even though strictly speaking, it is a mixture of
neurons, NSCs and also some astrocytes and sometimes
2.1. Materials oligodendrocytes. We call these neurons, e.g., “d10
Cell culture media, supplements, and hydrogels were neurons,” for 10 days of pre-differentiation. Additionally,
purchased from Fisher Scientific GmbH (Schwerte, we use the notation “(day A pp, diff B/C),” which reads
Germany). Cell culture flasks and plates were obtained “cells assessed A days post-printing with B days of pre-
from TPP-Techno Plastic Products AG (Trasadingen, differentiation and C days of post-differentiation.”
Switzerland). Chemicals and antibodies were purchased
from Sigma-Aldrich (Deisenhofen, Germany), unless 2.1.2. Cell culture media
otherwise noted. In the cell culture of this study, four different cell culture
media were used, a plating medium to initially seed the
2.1.1. Cells NSCs after thawing, an expansion medium to proliferate
All cells used were NSCs derived from hiPSCs or the NSCs, a neuronal differentiation medium to stimulate
differentiated from these NSCs under neuronal or the differentiation of the NSCs into neurons, and an
astrocytic culture conditions. This produced mixtures astrocytic differentiation medium to stimulate the
of cells from different differentiation stages. After 10 days differentiation of the NSCs into astrocytes. Immediately
of neuronal differentiation, the majority of the cells showed after the cells were passaged or printed, medium with
markers of mature neurons, but there were also NSCs that added RevitaCell™ was used. RevitaCell™ is a modified
were not yet differentiated. In addition, some cells did Rho-kinase inhibitor that promotes adhesion of neuronal
also spontaneously differentiate in a different direction. cells and prevents cell death. After 1 day, the medium was
For example, a few astrocytes were also found in neuronal replaced by medium without RevitaCell™.
differentiated cell cultures.
In this study, NSCs were printed without prior 2.2. Cell culture
differentiation, after neuronal differentiation for 5, 10, or 2.2.1. Stem cell culture
20 days, or after astrocytic differentiation. To avoid repetitive hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd.,
long descriptions, we will hereafter refer “NSCs” to as cells Cambridge, UK) were used in this study. The NSCs were
that have not been cultured in differentiation medium thawed and cultured as instructed by the supplier. Briefly,
-2
4
before the time of printing as NSCs, NSCs that have been NSCs were seeded at a density of 5 × 10 cells cm in Neural
in astrocytic differentiation culture as astrocytes, and NSCs Plating-XF Medium (Axol Biosciences) onto SureBond
Volume 9 Issue 2 (2023) 346 https://doi.org/10.18063/ijb.v9i2.672