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A novel 3D printing method for cell alignment and differentiation

muscle cells from secretory to contractile pheno- deposited, the printed trace spreads out due to its low
type [8,9] . However, when using multi- or pluripotent viscosity (at approximately 0.94 cP) [22] . The bioink
cells, the effect can aid the routing of cell differentia- consists of a biocompatible polymer, at a concentra-
tion towards desired cell types [7,10–12] . tion that confers for a viscosity able to retard the col-
Such exploitation of cell orientation by a modified lapse and spreading of the printed trace. However, the
surface has been defined as “Topographic guidance” concentration must not be too high, as not to hamper
or “Contact guidance” [12–14] . With regards to regenera- the sedimentation of the cells onto the etched grooves.
tive bioengineering, alignment of cells and extracellu- The hydrogel bioink chosen here was 2% gelatin in
lar matrix is a structural feature of several tissues and Dulbecco's modified eagle's medium, for which we
organs such as corneal stroma, vasculature, ten- have recorded a viscosity greater than 200 cP (data not
dons, bones, and skeletal muscles. It may also be use- shown).
ful in the culture and study of neurons [14,15] . Many cell types can be orientated and influe-
As mentioned, the patterned cues can affect the nced by such topographical guidance [4,14] . In this study,
phenotype of seeded cells. For example, mesenchymal we show the orientation and elongation of both fi-
stem cells (MSCs) seeded on electrospun-aligned silk broblasts and MSCs by the robot-etched grooves. As
fibroin cast on a rotating mandrel demonstrated en- an example of the phenotypic modulation induced by
hanced differentiation into ligament fibroblasts [15,16] . grooves, we demonstrate evidence of MSC differen-
Jiang et al. found that the seeding of human MSCs tiation towards cardiomyocytes, following elongated
(hMSCs) on aligned polycaprolactone fibers enhanced alignment on the etched surface [7,18,23,24] . Furthermore,
the differentiation towards neuronal lineage in the we assess whether cells bioprinted with the dispenser
presence of the induction factor retinoic acid [17] . Fi- are able to sense the grooves and alter their shape to
nally, MSCs have been found to respond to aligning respond to them. Hence, we aim to demonstrate a dual
features alone to differentiate towards a cardiomyo- role for the bioprinting apparatus to:
cyte lineage. Li et al. created aligned channels by di-  Be used for the modification of the cell seeding
rect femto-laser machining of poly(l-lactic acid)-co- surface by etching topographical guidance chan-
(ε-caprolactone) (PLLA-PCL) electrospun fibrous mats, nels,
and subsequently hMSCs were cultured onto these  Subsequent bioprinting of MSCs onto the guid-
scaffolds [18] . The cells elongated and aligned in the ing features in a gelatin bioink that allows the
direction of the channel, and also displayed signs of cells to sense the grooves,
lineage commitment towards cardiomyocytes, as dem-  The dispenser will use identically programmed
onstrated by quantitative Real-time polymerase chain co-ordinates for the etching and dispensing, so
reaction (qRT-PCR) [18] . that the cells are placed onto the grooves,
The surface modification through the introduction
of cell aligning channels or grooves is widely known  The viscosity of the bioink should contain the
as an effective method for the topographic control of cells’ spread for discrete patterns of cellularization.
cells. Cells have been found to align on grooves as This dual application of the dispenser system has
shallow as 150 nm [19] ; however, the deeper the groove, the potential to increase its usefulness for the produc-
the more effective they are for cell alignment, with the tion of more complex cell arrangements for scaffold
effect being noticeable down to depths of approxi- design in bioengineering.
mately 25 µm [20] . The reverse is true for channel width; 2. Material and Methods
as the width increases, the degree of cellular align-
ment decreases [9,21] . 2.1 Materials
Cells can be directly seeded onto a patterned sur-
face; however, when more than 1 cell lines are seeded Gelatin from the porcine skin (Bloom 300, type A)
and their spatial distribution is important, then the and phosphate buffer saline (PBS) of pH 7.2 were pur-
deposition of the cells require more accurate control. chased from Sigma-Aldrich. Human dermal fibroblasts
We have previously demonstrated how cells can be bio- (HDF) and human mesenchymal stem cells (hMSC)
printed with relative precision using robotic dispens- were supplied by Lonza, and red fluorescent protein
ing system, delivering viable cells within a hydro- expressing the rat mesenchymal stem cells (RFP-MSC)
[1]
gel bioink . When cell-containing growth medium is were supplied by Cyagen Biosciences Incorporation.
58 International Journal of Bioprinting (2015)–Volume 1, Issue 1