RESEARCH ARTICLE

Software tools for cell culture-related 3D

printed structures
Marton Gulyas1, Miklos Csiszer1, Elod Mehes1, Andras Czirok1,2*
1 Department of Biological Physics, Eotvos University, Budapest, Hungary, 2 Department of Anatomy & Cell
Biology, University of Kansas Medical Center, Kansas City, KS, United States of America

* aczirok@gmail.com

Abstract
a1111111111 Three-dimensional (3D) printing technology allowed fast and cheap prototype fabrication in
a1111111111
a1111111111 numerous segments of industry and it also became an increasingly versatile experimental
a1111111111 platform in life sciences. Yet, general purpose software tools to control printer hardware are
a1111111111 often suboptimal for bioprinting applications. Here we report a package of open source soft-
ware tools that we developed specifically to meet bioprinting requirements: Machine move-
ments can be (i) precisely specified using high level programming languages, and (ii) easily
distributed across a batch of tissue culture dishes. To demonstrate the utility of the reported
OPEN ACCESS technique, we present custom fabricated, biocompatible 3D-printed plastic structures that
Citation: Gulyas M, Csiszer M, Mehes E, Czirok A can control cell spreading area or medium volume, and exhibit excellent optical properties
(2018) Software tools for cell culture-related 3D even at 50 ul sample volumes. We expect our software tools to be helpful not only to manu-
printed structures. PLoS ONE 13(9): e0203203. facture customized in vitro experimental chambers, but for applications involving printing
https://doi.org/10.1371/journal.pone.0203203
cells and extracellular matrices as well.
Editor: Marco Livesu, Consiglio Nazionale delle
Ricerche, ITALY

Received: April 9, 2018

Accepted: August 16, 2018

Published: September 4, 2018 1 Introduction

Copyright: © 2018 Gulyas et al. This is an open The recent spread of 3D printing technology allowed fast and cheap prototype fabrication in
access article distributed under the terms of the numerous segments of industry and it also became an increasingly versatile experimental plat-
Creative Commons Attribution License, which
form in life sciences. Bioprinting now is efficient and accurate to build in vitro tissue models
permits unrestricted use, distribution, and
reproduction in any medium, provided the original with the potential to provide pathologically relevant responses and thus model human disease
author and source are credited. mechanisms. Bioprinted structures increasingly yield phenotypic endpoints that are compara-
ble with clinical studies and can provide a realistic prediction of clinical efficacy [1]. Several
Data Availability Statement: All relevant data are
within the paper, its Supporting Information files excellent papers review the various materials and bioprinting systems as well as their promise
and the http://petriprinter.elte.hu website. of clinical applications [2–6].
As a relevant example, a recent study describes bioprinting of three dimensional, cell-laden,
Funding: This work was supported through the
New National Excellence Program of the Ministry of
vascularized tissues that exceed 1 cm in thickness [7]. These constructs could be perfused on a
National Resources (UNKP-17-3-I-ELTE-32) and microfluidic chip for long time periods exceeding six weeks. Remarkably, the device can inte-
the Hungarian Scientific Research Fund (118119- grate up to three tissue types (parenchyma, stroma, and endothelium)—differentiated from
ANN). human mesenchymal stem cells (hMSCs) in a customized extracellular matrix environment.
Competing interests: The authors have declared In a similar effort, an artificial vascular network was reported by 3D printing of rigid sugar fila-
that no competing interests exist. ment networks, followed by embedding in a fibrin hydrogel. After dissolving the sugar, the

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 Software tools for cell culture-related 3D printed structures

resulting tunels can be populated with endothelial cells and perfused with blood under high-
pressure pulsatile flow [8]. Using a novel coaxial printing method a functional blood vessel can
be fabricated, where the lumen is filled with a water-soluble material, while the bio-ink for the
wall is a composite of ECM proteins and endothelial progenitor cells [9]. The structure of the
extracellular environment can be also shaped by 3D printing technology. As an important
example, follicle explants can be cultured in a suitable gelatin mesh structure, and can be used
as a functional ovarian implant in surgically sterilized mice [10]. Such implanted, follicle-
seeded scaffolds become highly vascularized and ovarian function was fully restored: pups are
born through natural mating and thrive through maternal lactation.
Affordable 3D-printing also allows the development of specialized devices for in vitro cell
technology. As an example, 3D printed inserts can be used to grow and stimulate neurons
within geometrically constrained compartments [11]. By fabricating structures in culture
dishes, one can control cell spreading, hence local cell density, or by restricting medium vol-
ume, decrease the amount of necessary reagents. 3D-printing technology also allows a simple
in-lab fabrication of channels and reservoirs in cell culture dishes—on a cruder scale than lito-
graphy-based microfluidic chambers, but without requiring specialized equipment and at a
fraction of the cost [12, 13]. While 3D-printers, with small modifications, are capable bioengi-
neering tools, these applications also require software tools that have distinct requirements
from general-purpose slicer applications that convert a 3D solid object into a sequence of
machine movements, conventionally encoded in g-code [14]. Most importantly, bioengineer-
ing applications like cell printing or fabricating cell-scale environments often require well-
defined, specialized motion patterns and an ability to reproduce it in parallel targets, like an
array of culture dishes. Prescribing each machine movement by manually editing the low-level
g-code sequence is laborious, error-prone and time-consuming. For this reason we developed
a software package which can represent machine movements or g-code elements by simple
functions of a high level programming language such as python or C#. We also created a
graphic user interface, PetriPrinter, which distributes the programmatically defined printer
movements into several culture dishes organized in a grid pattern.

2 Materials and methods

2.1 Cell lines and culture conditions
P31 cells were a kind gift from Prof. K. Grankvist (University of Umea, Sweden). 3T3 cells
were obtained from American Type Culture Collection (ATCC; CCL-92). Primary hippocam-
pal culture was a kind gift of Katalin Schlett and Krisztian Tarnok (Eötvös University, Hun-
gary), and was established according to the animal welfare permit PEI/001/1108-4/2013 and
PEI/001/1109-4/2013, in agreement with European Union and Hungarian legislation.
Cells were grown at 37˚C in a humidified, 5% CO2, 95% air atmosphere. For the cell lines,
Dulbecco’s Modified Eagle Medium (DMEM, Lonza) containing L-glutamine was supple-
mented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin-amphotericin
B (Lonza). Primary cultures of embryonic hippocampal cells were prepared from CD1 mice
on embryonic day 17/18 according to previously published protocol [15] using Neurobasal
(NB) medium supplemented with B27 (Invitrogen) containing 5% fetal calf serum (FCS,
Sigma), 0.5 μM GlutaMAX (Invitrogen), 40 μg/ml gentamicin (Hungaropharma, Budapest,
Hungary), and 2.5 μg/ml amphotericin B (Sigma).

2.2 Cytotoxicity screening

Cytotoxicity was evaluated by measuring the cellular protein content according to previously
published protocol [16]. Cells were plated in the inner 40 wells of a 96-well plate. After 48 h

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 Software tools for cell culture-related 3D printed structures

exposure to various amounts of polylactic acid (PLA), cell monolayers were fixed with 10% tri-
chloroacetic acid and stained for 15 min with sulforhodamine B (SRB). Cells were repeatedly
washed with 1% (v/v) acetic acid to remove excess dye. The protein-bound dye was dissolved
in 10 mM Tris and the optical density of the sample was determined at 570 nm using a micro-
plate reader (EL800, BioTec Instruments, Winooski, VT, USA).

2.3 Live/dead assay

To characterize overall cell culture health, the Live/dead viability/cytotoxicity Kit (Invitrogen)
was used according to manufacturer’s protocol. Briefly, live cells were labeled with 4 μM cal-
cein-AM in cell culture medium (DMEM supplemented with 10% fetal bovine serum) at 37˚C
for 30 minutes. Subsequently, dead cells were labeled with 8 μM ethidium homodimer-1 in cell
culture medium at 37˚C for 30 minutes. Cells were fixed with 4% paraformaldehyde in phos-
phate buffered saline.

2.4 Microscopy imaging

All images were made on a Zeiss Axio Observer Z1 inverted microscope with 10x Plan-Neo-
fluar or 5x EC Plan-Neofluar objective. The microscope was equipped with a Zeiss Axiocam
MRM CCD camera.

2.5 3D printing into Petri dishes

A fused fabrication filament-based machine (Ultimaker Original) was used for printing in Petri
dishes (for the detailed protocol see http://dx.doi.org/10.17504/protocols.io.rpfd5jn). We used
polylactic acid (PLA) filaments manufactured by Verbatim (product number: 55283, color:
gray, diameter: 2.85 mm). Printing temperature was 230 ˚C, nozzle diameter was 0.4 mm, print-
ing speed was 300-500 mm/minute, extruding rate was 0.027-0.041. For printing, 35 mm tissue
culture grade Petri dishes were used (Greiner, Cellstar TC cell culture dish, item no.: 627160).

2.6 Sterilization
3D printing in Petri dishes took place in open environment. After the printing process dishes
were sterilized with 70% (v/v) ethanol for 10 min then dried for 2 h under a sterile hood.

2.7 Quantitative analysis of image contrast

To measure the quality of phase contrast images, we utilized the Fiji programming environ-
ment [17]. First, wells were selected by an ellipse ROI (region of interest) applied to a compos-
ite mosaic image, built from multiple adjacent field of views. To detect the local brightness
changes, images were filtered through a Sobel edge detector. Cells were identified by a suitable
threshold value applied to the Sobel-filtered image. The Sobel filter values were then averaged
over pixels belonging to cells yielding a score for each image.

3 Results and discussion

3.1 Software tools
Our software consist of an application programming interface (API) and two graphic user
interface applications (Fig 1). As described in more detail below, the API (gCodeAPI.NET)
provides a convenient function library to represent g-code objects. The gCode Editor
application visualizes g-code from third party slicer applications and allows their manual mod-
ification and optimization for cell culture applications. Finally, the PetriPrinter application

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 Software tools for cell culture-related 3D printed structures

Fig 1. Overview of the various software tools and their applications. Geometrical objects can be encoded as python or C# files, as general use Standard Triangle
Language (.stl) files, or as a sequence of machine specific commands (g-code). Internally, extrusion and movement commands are represented as a binarized
gCodeCollection objects (.ptf) that can be edited and distributed into a number of parallel Petri dishes.
https://doi.org/10.1371/journal.pone.0203203.g001

distributes g-code objects into several culture dishes for batch printing. All parts of this soft-
ware package require at least a Windows XP system and a Microsoft.NET Framework 4 pack-
age, and are licensed under the Apache License 2.0.
3.1.1 gCodeAPI.NET. Native computer numerical control (CNC) programming language
(g-code) is a very limited programming language which does not contain variables or logical
operators and it is inconvenient to read by humans. This API encapsulates g-code commands
into functions of high-level programming languages such as C# or python (see code examples
in S1 Appendix). Besides the availability of the entire repertoire (variables, loops, flow control)
of these languages, gCodeAPI.NET adds easily interpretable commands such as line, arc, rela-
tive and absolute position, speed, extrude or temperature. Documentation of the command
library as well as code tutorials are available at the http://petriprinter.elte.hu website and on
github (http://github.com/gulyasmarton/PetriPrinter). As demonstrated in the S1 Appendix,
one can define a simple object like a cylinder surface with a few lines of code.
3.1.2 gCode Editor. Generic purpose CAD programs allow the design of complex struc-
tures in a more convenient way than building it up from the elements of our g-code API. Slicer
programs, which generate g-codes from a 3D solid model, do not offer a detailed control over

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 Software tools for cell culture-related 3D printed structures

the tool movements. When printing delicate structures, however, some extruder head move-
ments may pull thin, undesired PLA threads and deposit them on the tissue culture surface.
Close proximity of the hot extruder head and the tissue culture surface during head travel
can also deteriorate the surface. Thus, there is a need to re-route such movements away from
regions where cells are expected to adhere. Finding such problematic machine movements in
the raw g-code, however, is quite difficult.
To make such tasks easier, our gCode Editor combines features of printing path viewers
and text editors: One can navigate among the g-code commands while a parser function trans-
lates the code to human readable format and the Path Viewer draws the printing path. Using
this visual information and GUI tools, one can identify and replace the problematic head
movements: travel paths can be augmented by inserting new control points and existing con-
trol points can be relocated by mouse clicks. The output of the editor is an API object which is
compatible with the PetriPrinter program.
3.1.3 PetriPrinter. The PetriPrinter application is a g-code generator program which han-
dles model files and aligns them into suitable positions to be printed into culture dishes. Using
a graphic user interface one can assign an arbitrary combination of distinct objects to the avail-
able positions. The software provides collision-safe and optimized entry, exit and movement
between the dishes.
Simple PLA-printable dish positioning objects like grids and rings are distributed as exam-
ples with the program package. The application can be adjusted to various hardware platforms
by specifying settings such as printing temperature, start height, and row/column distances.
When using dish holders, determining the heights of culture surfaces is crucial to obtain
reliable prints. According to our measurments, the average print-by-print variation of the cul-
ture dish surfaces is 0.2 mm (15 wells, 8 independent runs), larger than the height of a single
layer. Thus, we augmented the printer with a microswitch probe to measure the surface height
of each dish (see below), and utilize this information by the PetriPrinter software.
3.1.4 Validation. To test if the gCodeAPI commands are translated into correct g-Codes,
we translated test objects utilizing the basic elements of the API such as lines and ellipses. We
extracted the coordinates from the generated g-Codes, and determined the distance between
the points and the corresponding mathematical curve. For each object we found the distance
between the g-Code points and the mathematical curve to fall wihin the numerical precision
limit (10−8 mm).

3.2 Printing PLA objects in tissue culture dishes

3.2.1 Modification of printer hardware. To print into tissue culture dishes, we modified
a hobby-level 3D printer (Ultimaker Original, Ultimaker B.V., The Netherlands). The fan
shroud was changed to increase the rate of air flow and to allow an air gap between the top of
the dish wall and the bottom of the shroud. The heater block was replaced by one with a cylin-
drical neck above the nozzle socket, in order to fit into culture dishes and to increase the print-
able area within the dishes (Fig 2).
To keep the tissue culture dishes at a pre-determined grid, one can either attach PLA-
printed sockets to the build plate (object submitted to thingiverse upon acceptance), or replace
the build plate with a dedicated stage where machined sockets are lined with O-rings to keep
the dishes in place. In this latter approach the z coordinate of each dish is determined by a ded-
icated probe with a microswitch (Fig 2). This approach is precise (repeatable) within the reso-
lution of the printer’s z axis positioning (12 μm).
3.2.2 PLA wells can provide optimal phase contrast optics. PLA wells can be shaped
in such a way that minimizes the deterioration of phase contrast optics by eliminating the

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 Software tools for cell culture-related 3D printed structures

Fig 2. Adaptation of an ultimaker original printer for culture dish printing. A: Stage accepting an array of 35 mm cell culture dishes. B: Modified printhead. Yellow
area: modified shroud to increase clearance to the stage and improve airflow. Blue area: a heater block that fits into the tissue culture dish. Red area: holder for a
removable microswitch sensor, used to map the height of each dish. C: Printhead with microswitch sensor installed (orange). Another microswitch sensor, mounted
on the stage (dark red) is used to calculate the height difference between the removable sensor and the nozzle.
https://doi.org/10.1371/journal.pone.0203203.g002

curvature of the medium interface. If wells have an appropriate conical shape, the equilibrium
contact angle between the wall and the medium is achieved by a flat medium-air interface.
To determine the suitable slope angle of the conical surface, we made a series of models
with angles ranging from vertical to outward leaning cones with an aperture angle of 100˚. As
Fig 3 demonstrates, for DMEM within a PLA container the phase contrast quality gradually
improves until the aperture of the cone reaches 80˚, i.e. the slope of the walls is 40 ˚. This
improvement reflects both a decreasing curvature of the medium surface and an increasing
distance of the medium-wall contact area.
3.2.3 PLA structures in tissue culture dishes. We demonstrate the utility of the approach
by some simple 3D printed structures in 35 mm culture dishes that are useful in various exper-
imental studies. Such dishes are convenient for time-lapse studies as they offer better phase
contrast images than 24 or 96 well plates.
Dish 1 (Fig 4A) allows to maintain seven independent cultures within the same 35 mm
dish, without sharing media and without deterioration of optical quality. Each well consists of
a 3 mm tall cylindrical wall, which expands into a conical surface of 1 mm high with the previ-
ously determined apex angle of 100˚. Dish 2 (Fig 4B) contains a small ring to limit the spread-
ing of cells and a higher, larger diameter outer wall to set the volume of medium within the
same 35 mm dish. Limiting cell spreading to a certain area can be used to locally set cell den-
sity. In particular, high cell densities can be maintained with a high medium volume/cell ratio,
enabling long term observation of cultures [18]. Dish 3 (Fig 4C and 4D) contains 1 mm2 mini-
wells that can be used to isolate cell clones, possibly obtained with a single cell sorter [19]. A
microscopic image of the single layer PLA wall indicates the resolution limits obtainable with
such technology: the 0.4 mm wide extrusion nozzle yields 120 μm tall and 920±90 μm wide
PLA barriers on the tissue culture plastic. The mean area between the barriers was determined
to be 1.82 mm2, while the standard deviation of individual cell areas was found to be 1.6.
3.2.4 Biocompatibility of PLA structures. To test the acute (short-term) cytotoxicity of
fusion-deposited PLA structures, we exposed two well-established cell lines, 3T3 fibroblasts
and p31 mesothelioma tumor cells, to various amounts of PLA. After two days in culture, the
total protein content of the cells, as a viability score, was determined by the SRB assay. As Fig 5
indicates, the viability of the cells was not affected by the presence of PLA.
Long term cytotoxicity was assayed using a primary hippocampal neuron culture (Fig 6).
Cells were seeded onto Poly-L-lysine-coated surfaces at a density of 900 cells/mm2. The

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 Software tools for cell culture-related 3D printed structures

Fig 3. Optimization of conical wells for phase contrast optics. A: A series of wells were printed with various wall
slopes or cone aperture angles (left column). 3T3 cells within the wells were imaged using an automated phase contrast
microscope. The tiled mosaic fields are shown in the middle column (scale bar: 500 μm). Red rectangles mark
individual micrographs, that are shown in the right column (scale bar: 100 μm). The best optical image corresponds to
a DMEM-PLA contact angle of 40˚. B: Quantitative analysis of image contrast. For each well we assigned a contrast

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 Software tools for cell culture-related 3D printed structures

quality value, and data is pooled from n = 3 parallel sets of wells. Asterisks denote significant (p < 0.05) difference from
the image obtained in cylindrical wells, error bars indicate standard deviation.
https://doi.org/10.1371/journal.pone.0203203.g003

Fig 4. Various 3D printed culture dish configurations. A: Seven-well dish with a sloped wall to provide optimal optical quality. B: A double ring dish to control cell
spreading and medium volume. C: A grid with 38 rectangular cells to store manually sorted cells. D: Phase contrast micrograph of a single PLA layer adhering to a
tissue culture substrate, part of the cell sorter grid shown in panel C. Air bubbles indicate small areas with imperfect contact between the PLA layer and the underlying
substrate. Scale bar: 500 μm.
https://doi.org/10.1371/journal.pone.0203203.g004

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 Software tools for cell culture-related 3D printed structures

Fig 5. Cytotoxity assay for PLA exposure. Cells were grown in the presence of PLA surfaces for two days after which
their protein content was determined by an SRB assay. Data are shown as average of n = 4 parallel experiments for two
cell ines (A: 3T3 cells, B: p31 cells) and the effect of treatment is expressed relative to untreated controls. Error bars
represent standard deviation.
https://doi.org/10.1371/journal.pone.0203203.g005

Fig 6. Long-term biocompatibility assay with primary hippocampal neuron culture. A: A triple ring dish used for
cell culture. B: To achieve high cell density, cell spreading is restricted within the rings (diameter: 6 mm, height: 1.8
mm). A tiled image of phase contrast micrographs demonstrate the optical quality of the setup. Scalebar: 1000 μm. The
red rectangle marks a region shown in higher magnification in panel C. D: Composite fluorescent image to evaluate
cell death within the same region shown in panel C. Live cells are labelled by calcein (green), while dead cells are
labelled by ethidium (red) and marked by arrows.
https://doi.org/10.1371/journal.pone.0203203.g006

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culture dish contained three PLA rings to limit cell spreading and to maintain high medium/
cell ratio. These conditions are favorable for long-term cell culture. After 7 days in vitro, cell
viability was determined using a fluorescent live/dead staining kit, which indicated a cell death
rate of less than 2.2.

4 Conclusions
3D printing is becoming wide-spread in life sciences, either to provide cost-effective alterna-
tive to existing experimental devices, or to fabricate highly specialized structures that are
specifically designed to meet certain experimental requirements. To effectively transfer 3D
printing know-how, some uniformization—a common language—is required. Our approach
can represent a variety of objects by sufficient detail and precision, yet in a conceptionally
clearer form than a sequence of machine-specific low-level movement commands. We hope
that this platform will thus facilitate both the distribution as well as the development of new,
experiment-specific devices or in vitro cell technologies.

Supporting information
S1 Appendix. Example codes using the gCodeAPI. A cylinder surface is defined in C# (A) or
python (B) language.
(PDF)

Acknowledgments
We are grateful to Tamás Garay for his help with the SRB cytotoxicity assay, Krisztián Tárnok
and Katalin Schlett for their gift of the hippocampal neuron culture used in this study.

Author Contributions
Investigation: Marton Gulyas, Elod Mehes.
Methodology: Miklos Csiszer.
Software: Marton Gulyas.
Supervision: Andras Czirok.
Writing – original draft: Marton Gulyas, Andras Czirok.
Writing – review & editing: Elod Mehes, Andras Czirok.

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