New BIOTECHNOLOGY 39 (2017) 222–231

Contents lists available at ScienceDirect

New BIOTECHNOLOGY
journal homepage: www.elsevier.com/locate/nbt

Review article

Additive Biotech—Chances, challenges, and recent applications of additive

manufacturing technologies in biotechnology
⁎
Felix Krujatza, , Anja Lodeb, Julia Seidela, Thomas Bleya, Michael Gelinskyb,
Juliane Steingroewera
a
Institute of Natural Materials Technology, TU Dresden, Bergstraße 120, 01069 Dresden, Germany
b
Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Fetscherstraße 74, 01307
Dresden, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The diversity and complexity of biotechnological applications are constantly increasing, with ever expanding
Additive manufacturing ranges of production hosts, cultivation conditions and measurement tasks. Consequently, many analytical and
3D printing cultivation systems for biotechnology and bioprocess engineering, such as microfluidic devices or bioreactors,
Bioprocess engineering are tailor-made to precisely satisfy the requirements of specific measurements or cultivation tasks. Additive
Biotechnology
manufacturing (AM) technologies offer the possibility of fabricating tailor-made 3D laboratory equipment di-
Microfluidics
Bioprinting
rectly from CAD designs with previously inaccessible levels of freedom in terms of structural complexity. This
review discusses the historical background of these technologies, their most promising current implementations
and the associated workflows, fabrication processes and material specifications, together with some of the major
challenges associated with using AM in biotechnology/bioprocess engineering. To illustrate the great potential of
AM, selected examples in microfluidic devices, 3D-bioprinting/biofabrication and bioprocess engineering are
highlighted.

Introduction Historical development

Terminology The history of 3D printing begins in 1983 in California, when the

The term additive manufacturing (AM) refers to a very wide range
of technologies: in 2010, it was defined by the American standardiza-
tion organization ASTM as “…a process of joining materials to make
objects from three-dimensional (3D) model data, usually layer upon
layer, as opposed to subtractive manufacturing methodologies…” [1]. It
is largely synonymous with several other terms that have been used in
the literature, including 3D-printing, rapid prototyping (RP), rapid
manufacturing (RM), direct digital manufacturing, solid freeform
techniques and layer manufacturing. Despite the very similar meanings
of these terms, it is important to differentiate between rapid proto-
typing (defined as “… a process for rapidly creating a system or part
representation before final release or commercialization…”) and rapid
manufacturing, which involves “…the use of AM to produce parts
which will be used as an end-product…“ [2].
American engineer Charles “Chuck” Hull developed and patented the
first AM device, which was used to manufacture 3D products by using
UV light to cure photopolymers. This technology and the corresponding
device were patented as “stereolithography” [3]. In 1986, Hull founded
the company 3D-Systems, which launched the first commercial avail-
able 3D Printer: the SLA‐1. Over the last two decades, several compa-
nies have followed in the footsteps of 3D-Systems, including Stratasis
from the US, Arcam from Sweden, and EOS from Germany. These
companies have developed innovative AM technologies and devices
that can work with a great variety of materials including ceramics,
composites and metals. While the first AM systems were better suited
for RP-type applications, advancements made by these firms and others
have driven a transition towards RM in research and industry.

Abbreviations: AM, additive manufacturing; PDMS, polydimethylsiloxane; CAD, computer-aided design; RP, rapid prototyping; RM, rapid manufacturing; NIR, near-infrared; LOC, lab-
on-chip; FPA, flat-panel-airlift; PBR, photobioreactor; OLED, organic light emitting diode; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
⁎
Corresponding author.
E-mail address: Felix.Krujatz@tu-dresden.de (F. Krujatz).

http://dx.doi.org/10.1016/j.nbt.2017.09.001

Available online 07 September 2017

1871-6784/ © 2017 Elsevier B.V. All rights reserved.
F. Krujatz et al.
Fig. 1. (A) CAD-model of a test structure; (B) surface geometry of the CAD-model expressed by 92 triangles (.stl-file, binary file of 4684 bytes); (C) fabricated test structures from
polyamide using the selective laser sintering technology and (D) from Accura Si60 using stereolithography.
Workflow of additive manufacturing
The first step in the fabrication of a 3D-object is to design a CAD-
based model, which is then converted into a .stl (standard tessellation
language or stereolithography) file. The .stl format describes the un-
structured surface geometry of the 3D-object in terms of the unit normal
and vertices of triangles using a 3D Cartesian coordinate system. This
file format was developed by Chuck Hull and some coworkers at 3D-
Systems during the late 80′s and remains one of the most important
interfaces between CAD software and AM devices today. The .stl file is
processed with a so-called “slicer software” that converts the geome-
trical object into thousands of 2D-layer data [4], which are translated
by the AM device to build up the 3D-object layer by layer (Fig. 1).
Several factors must be considered to identify the most suitable AM
technology for a given application, including the required object ac-
curacy, the object’s size, the material requirements and the cost of the
material [5]. Once the 3D-object has been fabricated, final finishing is
generally necessary. This may involve removing excess material or
supporting structures, polishing, lacquering, coloring, or infiltrating.
Materials & processes
AM technologies have the potential to create a new industrial re-
volution [6] while providing new degrees of freedom in terms of
structural complexity for (biotechnological) fabrication. Today, many
different plastics, metals, ceramics, polymer plasters and resins can be
processed using a wide range of technologies. The following section
focuses on widely used AM technologies that have the greatest potential
for use in biotechnological applications yet. Table 1 provides a detailed
overview of the materials used in these technologies as well as their
fabrication parameters and properties. From a bioprocess engineer’s
point of view, there are two particularly important parameters to
consider: the material’s heat stability in case heat sterilization is ne-
cessary and the required object accuracy (Table 1).
The 3DP/binder jetting (Fig. 2A) uses a powdery polymer plaster
that is distributed as a thin layer on a carrier plate using a roller system
[7]. The powder material is then hardened by integrating a binding
material via inkjet-print heads. The carrier plate is then lowered by
around 0.1 mm and the next layer of powder is distributed and fabri-
cated with the inkjet and so on until the complete 3D-object has been
built up. Finally, excess powder is removed and recycled for use in the
next fabrication process. In contrast, the Fused Deposition Modeling
(FDM) approach, also denoted Fused Filament Fabrication (FFF) in the
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New BIOTECHNOLOGY 39 (2017) 222–231
literature, developed by Stratasis, uses plastic materials, usually fila-
mentous acrylonitrile-butadiene-styrene (ABS), which is heated until
the material’s flow characteristics become suitable for extrusion
through a dosing nozzle (Fig. 2B). In addition to the building material,
FDM uses a second plastic to fabricate supporting structures, which
must be removed at the end of the fabrication process. Because of the
low heat stability of the fabricated materials and their currently limited
object accuracy, 3DP/binder jetting and FDM are typically used for RP-
applications.
The transition from RP (i.e. the creation of models) to RM (the
creation of practical components) was, for example, enabled by the
development of laser-based AM technologies such as Selective Laser
Sintering (SLS). SLS processes use plastics such as polyamides, elasto-
mers, nylon, or alumide that offer high mechanical stability, bio-
compatibility, high processing precision and low material costs [8]. The
SLS manufacturing process uses a powdery starting material that is
heated at elevated pressure on a carrier platform (Fig. 2E). The local
sintering of the material layers is induced with a focused laser beam,
typically a CO2 or Nd:YAG laser, and the carrier is lowered in a stepwise
fashion to enable the layer-by-layer construction of the object [9]. As
with 3DP, the final step in the manufacturing process is to remove
excess powder.
Selective Laser Melting (SLM) and Electron Beam Melting (EBM)
permit fabrication with metallic powder materials such as tool steel,
stainless steel, titanium or aluminum [10,11]. To prevent corrosion,
SLM and EBM are performed under a protective atmosphere (Fig. 2C,
D). The metallic source materials and the requirements of the fabrica-
tion process make metallic AM comparable expensive. However, SLM
and EBM enable the production of highly complex, non-porous and
sterilizable 3D-objects with excellent heat stability and mechanical
properties [12,13].
The stereolithography (SLA) process uses UV-curable photo-
polymers, elastomers, epoxies or acrylates [14]. The liquid photo-
polymer is placed into a bath with a retractable carrier plate and is then
locally hardened by means of a mirror-controlled UV-laser (Fig. 2F).
The carrier plate is then lowered in a stepwise fashion to build up the
3D-object layer-by-layer. The use of a liquid source material necessi-
tates the use of additional supporting structures to fix the component
within the water bath, which must be removed after the fabrication
step. SLA-fabricated components have high levels of detail accuracy and
very favorable mechanical properties. In addition, the SLA process en-
ables the production of (semi)-transparent components.
The PolyJet™ (PJ) process closely resembles the MultiJet Modelling
F. Krujatz et al. New BIOTECHNOLOGY 39 (2017) 222–231

Table 1
Overview of AM technologies, materials, fabrication parameters and manufacturers of AM devices. Parameters are current benchmarks and were averaged from several manufacturer and
literature data. Indeed, they are continuously changing and improved by new technology developments.

Technology Source material Available materials Layer thickness Temperature stability Companies
[μm] [°C]

Stereolithography (SLA) Liquid-photopolymers Elastomers, Epoxy resins (e.g. Accura) 3D Systems

+ Micro-SLA
Ceramics Acrylates Autdesk
Ceramic particles in liquid resins (e.g. silica, 50–250 80 Digital Wax Systems
alumina, zirconia)
EnvisionTEC
Rapidshape
PolyJet™ (PJ) Acryl-photopolymers VeroClear, VeroWhite, TangoBlack (rubber- 16–32 50 Stratasys
like), AR-family
Digital ABS
Multi-jet modeling (MJM) Acryl-photopolymers VisiJet® family (polycarbonat-like) 16–32 50 3D Systems
Stratasys
Solidscape
3D-Printing (3DP)/Binder Chalky powder Plaster, wax, acrylic plastic 3D Systems
Jetting
Ceramics Zirconia, silica, sands, Ti3SiC2 150–500 115 Voxeljet
Composites Ceramic-ceramic, polymer-ceramic
Fused Deposition Modeling Plastic filament Acrylnitril-Budadien-Styrol (ABS) 100–1000 80 Stratasys
(FDM)
Composites Polylactids, waxes MakerBot
RepRap
Fabbster
Fused Filament Fabrication Plastic filament Polyether ether ketones (PEEK) 100–1000 260 Apium Additive
(FFM) Technologies
Electron Beam Melting (EBM) Metal powder Titanium 50–100 500–1000 Arcam
Cobalt-Chrome FIT Group
Sciaky
Selective Laser Sintering (SLS) Plastic powder Polyamid (e.g. PA2200), Polystyrene 100–200 80–150 3D Systems
Thermoplastic polyurethane (TPU) EOS
Ceramic powder Bioglass, silica,zirconia, alumina, sands, Blueprinter
graphite
Composites Composite Materials (e.g. Alumide, ceramic- 3Geometry Matsuura
ceramic, polymer-matrix materials)
Selective Laser Melting (SLM) Metal powder Stainless Steel, Tool Steel, Aluminium, 20–200 350–1000 EOS
Titanium, cobalt-chrome
SLM Solutions
Renishaw
®
2-Photon-Polymerization Photoresists SU-8, OrmoComp < 1 μm 100–300 Nanoscribe

(MJM) process and combines the production characteristics of inkjet-
printing with the advantageous properties of photopolymers (Fig. 2G).
Because PJ uses inkjet printing heads, the UV-curable source material
must be available in a viscous form to enable printing. The building
material print-head must be paired with a second print-head that is
used to produce supporting structures. The components fabricated by
the inkjet are usually cured directly after fabrication by treatment with
a UV-laser. The resins used in these processes (e.g. VeroClear) provide
high levels of detail accuracy [15] and mechanical robustness. The
material properties of the finished object can be tuned by performing
additional post-treatment steps (e.g. painting or polishing) or by using
more than two inkjet heads.
The AM technique that offers the highest object resolution for 3D
objects is 2-Photon-Polymerization of photoresists, which relies on a
non-linear two-photon absorption process [16]. This technology uses
high-power, femtosecond lasers that emit photons in the near infrared
spectral range. If two NIR-photons are absorbed simultaneously, the
power input corresponds to the absorption of one UV-photon, inducing
the same sort of polymerization reaction as is used in the PJ or SLA
processes. However, the NIR femtosecond pulses can be focused on a
very small volume known as a voxel (a 3D-pixel), causing tightly lo-
calized initiation of the polymerization reaction.
While the AM technologies discussed above are the most widely
used today, several others have been developed including selective heat
sintering, robocasting, laminated object manufacturing, ultrasonic
consolidation, direct ink writing, and contour crafting. These techni-
ques are discussed in detail elsewhere [2,17,18].
Applications of AM technologies in biotechnology
AM-fabricated microfluidic devices
One of the most popular and extensively reviewed applications of
AM technologies is in the fabrication of task-specific microfluidic de-
vices [19–22]. Microfluidic devices are used to handle and analyze
fluids on scales of μL- to mL, and are typically referred to as Lab-on-
Chip (LOC) or miniaturized total analysis systems (μTASs) [22]. They
have numerous uses in biomedical [23], environmental [24], analytical
[25], chemical [25], biotechnological [26] and point-of-care applica-
tions [27]. A microfluidic device for analyzing cellular properties
should have good transparency, to facilitate optical monitoring of
bioprocesses, and high gas permeability. Most microfluidic devices
developed over the last 15 years were fabricated in glass or silicone by
lithography, or by soft lithography using PDMS. However, lithography
is a costly and time-consuming multistep process (partly because of the
need to fabricate molds) that makes it difficult to incorporate devices
that facilitate automation (e.g. pumps and valves) and is not readily
scaled up to produce large numbers of devices [28]. Unlike lithographic
approaches, AM technologies do not require molds and make it possible
to fabricate microfluidic devices in a single step, while offering full
creative design freedom in 3D space. The AM technologies that are used
most extensively to create microfluidic devices today are FDM/FFF,
PolyJet™, SLA, 2-PP and DMD-PP.
Bonyar and coworkers in 2010 combined AM technology with
conventional PDMS molding by fabricating a master mold from an

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F. Krujatz et al. New BIOTECHNOLOGY 39 (2017) 222–231

Fig. 2. Illustration of the workflow for different AM technologies; with kind permission from additively (https://www.additively.com/): (A) 3D-Printing/Binder Jetting; (B) Fused
Deposition Modeling (FDM)/Fused Filament Fabrication (FFF); (C) Electron Beam Melting (EBM); (D) Selective Laser Melting (SLM); (E) Selective Laser Sintering (SLS); (F)
Stereolithography (SLA); (G) PolyJet™/Multijet Modeling (MJM).

acrylic photopolymer with a PolyJet™ device [29]. These authors re-
ported the casting of a PDMS flow cell used in a Surface Plasmon Re-
sonance device and the creation of several functional elements such as
chaotic mixers and fluid homogenizers. Similar approaches involving
the use of AM technologies to fabricate master molds were described by
King et al. [30] and Spivey et al. [31]. The King group used the
PolyJet™ technology with materials from the VeroClear family to
fabricate a PDMS casting mold that was used to create interdroplet
bilayer arrays, which in turn were used to study membrane transport
phenomena. Spivey and coworkers used a multiphoton lithography
process to fabricate a cell-capture device. This system was then used to
study aging and protein aggregation in the fission yeast Schizosacchar-
omyces pombe.
Other groups have used AM technologies to fabricate microfluidic

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F. Krujatz et al.
Fig. 3. (A) 3D-designed microfluidic device for capturing bacteria by inertial focusing (MNC = magnet nanoparticle clusters); (B) Visualization of forces (FL = lift force, FD = dean drag
force) within microfluidic trapezoid channels; (C) AM-fabricated device by using a 3D Systems SLA system, pictures taken from reference 42 with kind permission from Nature Publishing
Group; (D) Microscopic images of a micro-textured microfluidic channel realized by two-photon polymerization, picture taken from reference 55 with kind permission from Springer.
devices with passive microfluidic elements such as droplet generators
[32,33], microcapillary emulsion generators [34], functional micro-
fluidic valves [35], chip interconnectors [36] or flow meters [37]. In
addition, the design and fabrication of active microfluidic components
such as pumps [19] and microlenses has been reported [38]. Notably,
Rogers et al. [39] used the SLA technology to fabricate fluidic devices
with integrated membrane-based valves from a custom-made resin.
However, the authors noted that the SLA-produced device exhibited
limited transparency and high autofluorescence, which hinders the
optical monitoring. Similar observations were made by Shallan et al.
[40], who performed SLA fabrication with a resin made of an acrylate
oligomer and monomer together with a photoinitiator and additives.
This material achieved a transparency of 60% in the region between
430 and 620 nm. Additionally, it exhibited no autofluorescence at an
excitation wavelength of 480 nm, enabling optical on-chip detection.
SLA was used together with the non-swelling and transparent resin
Somos WaterShed XC by Lee et al., who developed an immunomagnetic
flow assay based on antibody-functionalized magnetic nanoparticles for
capturing and quantifying Salmonella bacteria in water samples [41]
and E. coli from milk [42], respectively (Fig. 3A–C). Separately, a Czech
group used the FDM/FFF process with ABS or polylactic acid to fabri-
cate a number of similar bio-analytical microfluidic devices for de-
tecting the influenza virus [43], methicillin-resistant Staphylococcus
aureus [44], and nucleobases in hydrolyzed samples [45]. MacDonald
and coworkers [46] evaluated the biocompatibility of various materials
and AM fabrication processes (MJM, FDM/FFF and SLA) using the
zebrafish embryo toxicity assay. Their results indicated that most of the
untreated materials had dramatic negative effects on embryo survival.
To alleviate this problem, they also developed a pre-treatment protocol
that significantly increased the materials’ biocompatibility.
To fully realize the potential of AM, it would be desirable to es-
tablish integrated microfluidic design and fabrication processes in
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New BIOTECHNOLOGY 39 (2017) 222–231
which the capillary layout, integrated functional devices and sensing
components are all processed similarly [47,48]. Some steps in this
important direction have already been taken. For instance, the Mid-
orikawa group used a 2-PP femtosecond laser direct writing method to
fabricate microfluidic and microoptical components such as optical
waveguides and filters in photostructurable glass chips called nanoa-
quariums [49]. 2-PP enables highly accurate fabrication of nanometer-
scale structures [50] and has thus been used to construct a wide range
of nanoscale microfluidic components including nanofluidic filter ele-
ments [51], nozzles, magnetic microrobots [52,53], wall-less micro-
channels [54], and structures with surfaces having varied topographies
[55] (Fig. 3D). Digital micromirror device-based projection printing
(DMD-PP), a technology very similar to the SLA-process, is widely used
to create microenvironmental structures for studying cellular responses
to topographical conditions. It has been already used to construct mi-
mics of the extracellular matrix [56], scaffolds with selected cell-ad-
hesive sites [57], scaffold geometries with varying stretching properties
[58,59] and scaffolds with different pore structures [60].
Another important step towards the further commercialization of
AM technologies for microfluidic applications was taken by Dolomite
Microfluidics, which recently launched the Fluidic Factory − the first
commercial available AM device for fabricating tailor-made micro-
fluidics from a cyclic olefin co-polymer.
Biofabrication/3D-bioprinting for biotechnology applications
The terms biofabrication and 3D-bioprinting are very closely related
[61]. Here, they both refer to the simultaneous processing of living cells
and various biomaterials using AM technologies. Several AM technol-
ogies, including inkjet printing [62,63], extrusion-based printing [64]
and laser-assisted printing [65] can be used for this purpose [66]. Work
by pioneering researchers such as Landers and Mühlhaupt [67] has
F. Krujatz et al. New BIOTECHNOLOGY 39 (2017) 222–231

demonstrated that extrusion-based technologies such as 3D-plotting are
powerful tools for constructing volumetric 3D cell-laden hydrogel
constructs. The 3D-structure is generated by extruding a cell-laden,
highly viscous or pasty plotting material through the dosing needle of a
3D-plotting device. The needle’s movements are software-controlled,
allowing a 3D-structure to be built up layer-by-layer. Recent publica-
tions have demonstrated the high viability of structures with hydrogel-
embedded cells, fabricated using 3D-plotting including gels based on
alginate [68], agar [69], gelatin and their derivatives [70,71] and
composite materials [72,73].
The term “hydrogel” refers to a 3D hydrophilic network consisting
of natural or synthetic polymers with high water content [74]. The
properties of hydrogels are typically tailored to their applications,
which span a diverse range of fields including biomedicine [75], bio-
technology [76], the food industry [77], pharmacy [78], cosmetics
[79], and agricultural sciences [80]. While 3D-bioprinting of living cells
has been used in the field of tissue engineering for several years [66], its
applications in biotechnology remain largely unexplored. The challenge
of using 3D-bioprinting in biotechnological applications is to find pro-
cessing conditions for the hydrogel that allow the fabrication of 3D-
structures while maintaining a high survival rate among the embedded
cells [81]. One strategy to meet these requirements was developed by
Schütz and coworkers, who increased the viscosity of an alginate so-
lution temporarily by adding 3% methyl cellulose [82]. This yielded a
material whose mechanical properties were very well suited to the
extrusion process, allowing the fabrication of mechanically stable 50-
layer hydrogel structures. The methyl cellulose which is washed out
during the subsequent cultivation showed no toxic effects towards
embedded mesenchymal stem cells and produced a highly porous
structure at the micro-level, enabling an effective nutrient supply.
The same fabricated hydrogel (Fig. 4A) construct was successfully
used to immobilize the microalgal strains Chlamydomonas reinhardtii
11–32b [83] and Chlorella sorokiniana UTEX1230 [84] (Fig. 4B). Cel-
lular viability was monitored by fluorescence microscopy and a Fiji-
based image analysis, revealing that the shear forces generated during
the 3D-plotting process had no negative effect on the cells’ viability
[83]. The algal cells were homogenously distributed in the hydrogel
environment by the plotting process (Fig. 4B–E). Under cultivation
conditions that promote the photoautotrophic growth of C. reinhardtii
11–32b, the number of cells increased over 12 days of cultivation. In
addition., the immobilized cells formed highly viable and photo-
synthetically active cell clusters (30–40 μm), demonstrated by fluores-
cence microscopy using the autofluorescence of chlorophyll and by
measuring the concentration of photosynthetically generated oxygen in
the surrounding medium (Fig. 4E). As a proof of principle and to de-
monstrate potential (bio-) medical applications, the authors reported
the fabrication of patterned human/algal co-culture scaffolds by mul-
tichannel-plotting (Fig. 4F,G), which is the basis for new therapeutic
concepts that circumvent the oxygen limitations of human cell lines
during in vitro tissue engineering. This technology, which we have
named Green Bioprinting, could recently be expanded to plant in vitro
cultures [85] and is the basis for new biotechnological applications,
such as cascaded bioprocesses using the metabolic properties of dif-
ferent cell types, or to establish model systems for studying biofilms or
symbiotically living species. Similar studies on bacterial quorum sen-
sing were presented by Connell and coworkers [86,87], who used a 3D-
microprinting approach with gelatin and composites (Fig. 4H–K) to
fabricate structured bacterial 3D-micro communities with the Gram-
positive Staphlyococcus aureus and Gram-negative Pseudomonas aerugi-
nosa. These investigations revealed that the β-lactam antibiotic re-
sistance of P. aeruginosa is influenced by a quorum sensing metabolite
produced by S. aureus.
Next generation bioprinting approaches (“4D bioprinting”) are fo-
cusing on the combination of intelligent biomaterials that can change

Fig. 4. (A) Illustration of a 3D extruded alginate/methylcellulose 4-layer scaffold after the plotting process; (B) Chlamydomonas reinhardtii 11–32b (microalgae) cells were mixed within
the plotting paste and incubated under photoautotrophic conditions for 12 days, microalgal growth is indicated by an intensive green coloring of the hydrogel construct; (C) photo-
synthetic active cell clusters are visible within the hydrogel environment; (D) microscopic image of the 3D hydrogel layer structure; (E) local- and time resolved data of single cell cluster
development at the micro-level is accessible by microscopic image analysis; (F) CAD-model of a 3D patterned algae/human SaOS-2 co-culture (purple = SaOS-2, green = algae); (G)
brightfield image of an MTT-stained patterned co-culture (blue = SaOS-2, green = algae); (H) procedure of gelatin-bacteria-based micro-3D printing (red arrows = 37 °C, blue
arrows = 18–22 °C); (I) confocal fluorescence (cf) isosurfaces show isolated Pseudomonas aeruginosa microcolonies within a pyranoid; (J) cut out views of cf isosurfaces showing six
physically segregated P. aeruginosa populations; (K) brightfield Image 10 min post fabricated (left), side-view and partially top-down view on spheroidal chambers which are connected by
channels including P. aeruginosa cells (after 18 h of cultivation) (scale bars, 20 μm). Fig. 4H–K taken from reference 85 with kind permission from the Proceedings of the National
Academy of Sciences of the United States of America. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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their functionalities over time or in consequence of external stimuli
[88,89].
Additive manufacturing for advanced bioprocess engineering
As yet, AM technologies have not been widely used to fabricate
advanced functional laboratory-scale bioreactor components or culti-
vation equipment. Preliminary investigations into the production of
tailor-made labware for bioprocess engineering were reported by
Lücking and coworkers, who conducted a case study involving the de-
sign of a baffled microwell plate made from polyamide with a great
variety of different well geometries [90]. The SLS-fabricated plate was
tested in the cultivation of S. cerevisiae and both adherent and sus-
pended human cells [91]. In another case, a CAD-designed shake flask
cap equipped with different Luer-lock connectors was developed to
enable sampling without cap removal or feeding into the medium. Caps
were produced using FDM/FFF and SLS technologies, revealing that the
limited object accuracy of FDM/FFF was problematic in this case. In
contrast, the SLS-fabricated polyamide cap could be used directly after
fabrication without limitations. The usefulness of “smart” 3D-designed
shake flask caps was demonstrated by Ude et al. [92], who constructed
a “shake flask pH-controller unit” (SFC) (Fig. 5A). This semi-autarkic
“smart cap” includes material-integrated channels, membranes and
valves, as well as controlling electronics and piezoelectric micropumps,
to close the control loop by pumping NaOH or HCl into the culture
broth. The SFC was successfully used in a controlled shake flask culti-
vation of E. coli K12, during which it maintained a narrow pH range in
the culture medium.
Khan et al. presented a novel rocking bioreactor for fermentative
hydrogen production that features internal AM-fabricated porous car-
tridges made from polylactic acid with the REPRAP 3D printing plat-
form [93]. The cartridges were filled with immobilized microbial
communities and were designed to increase the cells’ exposure to the
culture medium by acting as baffles and facilitating mixing. The tailor-
made reactor system exhibited shorter lag time and a higher peak hy-
drogen production than a conventional production system. Kazenwadel
and co-workers developed a modular reactor setup consisting of porous
monolithic enzyme carriers made from VeroClear RGD810 (PolyJet®)
and an AM-fabricated housing with integrated fluid distributors [94].
The 3D-layout further included integrated temperature and pH reg-
ulation. The functionality of the modular enzyme reactor was demon-
strated by a two-step enzyme transformation including glucose oxidase
and horseradish peroxidase. The 3D-fabrication of porous columns and
monolithic structures could be also useful as an analytical system in
biotechnology, as exemplified Fee and coworkers [95].
The first completely AM-fabricated bioreactor system – the
MicrOLED-photobioreactor (PBR) – was recently described by our
group [96]. The MicrOLED-PBR is a miniaturized flat-panel airlift-PBR
(FPA-PBR) with an overall volume of 15 mL and several useful features
that were realized by integrating the design of its electronic systems
with that of the bioreactor as a whole during the 3D layout process
(Fig. 5B, C). The FPA-PBR was made from polyamide using the SLS
technology and includes a sensor board containing optical elements
(LEDs, photodiodes and control electronics) for non-invasively mon-
itoring cell-specific process parameters such as the cell density and
chlorophyll fluorescence. The concentrations of dO2 and dCO2 in the
medium as well as its pH were measured with optical sensor spots that
were read out using polymer optical fibers (POF) fixed inside a POF

Fig. 5. (A) CAD-model of the shake flask control unit (SFC) and functional components. The SFC is compatible with standard Corning shake flasks, photograph of the additive manu-
factured SFC from polyamide using selective laser sintering, picture take from reference 91 with kind permission from Elsevier; (B) left: housing of the MicrOLED-PBR made from alumide
using selective laser sintering, right: FPA cultivation chamber of the MicrOLED-PBR with tailor-made PBR lid from stainless steel using selective laser melting; (C) photograph of the
MicrOLED-PBR with combined electronic and structural design; (D) dual-plane external OLED illumination provides an ideal homogeneous light distribution within the FPA cultivation
chamber.

228
F. Krujatz et al.
holder embedded in the 3D polyamide structure. The photo-
synthetically active radiation in the reactor was supplied by organic
light emitting diodes (OLED). Therefore, a 3D-illumination frame de-
sign was adopted that permits dual-plane external illumination of the
scaled-down FPA-cultivation chamber by two integrated OLED-modules
(Fig. 5D). The MicrOLED-PBR enables rapid strain screening and small-
scale bioprocess development for photobiotechnological processes, and
represents the first step towards a new generation of tailor-made AM-
fabricated bioreactors designed to precisely satisfy the requirements of
specific cultivation processes.
Metal-processing technologies such as SLM or EBM could provide
considerable added value in bioprocess engineering, for instance in the
design and one-step fabrication of advanced microbioreactors with in-
tegrated functionalities. Fig. 5B depicts the use of SLM to fabricate a
structurally complex bioreactor lid with integrated aeration and sam-
pling pipes made from stainless steel for use with the MicrOLED-PBR in
continuous process operation. Metal-based AM systems could also en-
able the design and construction of many innovative task-specific
bioreactor stirrer systems. The potential of AM technologies for the
design of laboratory equipment and automation is further highlighted
in a series of recently published articles [97–99].
Chances & challenges
Obviously, there are many challenges and opportunities associated
with the use of AM technologies for tailor-made biotechnology and
bioprocess engineering. AM technologies have a number of important
advantages over conventional fabrication processes. First, because of
their additive nature, they produce notably less waste material than
subtractive manufacturing processes [2]. In addition, AM technologies
enable the fabrication of very complex and even “smart” structures that
could be useful in integrated bioreactor components such as sensors,
actuators or capillaries for temperature control [100]. A major ad-
vantage of AM methods is their short processing times, which range
from hours if a device is available on site to days if it is necessary to use
an online service. In biomedicine, 3D-data obtained using scanning
devices such as computerized tomography instruments, laser scanning
devices or magnetic resonance imaging facilities could be used directly
to fabricate patient-adapted scaffold constructs or implants [101].
As noted above, the selection of a suitable AM technology for a
given application should primarily be guided by the material require-
ments and the achievable resolution of the 3D-objects, which generally
varies from the nm- to the μm-scale. Due to the high cost of purchasing
an AM device, most users will only be able to access such machines by
making use of the online services provided by various companies, or by
using public FabLabs or MakerSpaces. Most AM processes require only a
single AM device and the size of the 3D-object will generally be limited
by the dimensions of the available devices. Another limitation on the
use of AM technologies is that while the main fabrication step may
proceed quickly, post-fabrication processes such as polishing often re-
quire time-consuming and costly manual work. Finally, it is important
to be aware that increasing the level of object accuracy or using ma-
terials with specific properties may increase production costs when
using AM methods.
Efforts to expand the limited range of materials usable in current
AM processes are ongoing in many different laboratories around the
world. In particular, there is great interest in identifying new composite
materials with useful properties that can be employed in AM [102,103].
There are also continuing efforts to improve the AM devices themselves.
In addition to new device designs [104], there are regular advances in
process control [105] and in sensor systems for process monitoring to
reduce process variability across different devices and over time
[2,106]. It would also be desirable to see a stronger focus on developing
multi-material processing devices [107] and technologies capable of
fabrication with renewable materials. A number of visionary ideas have
been proposed, such as AM fabrication of electronic components that
229
New BIOTECHNOLOGY 39 (2017) 222–231
could be used to create fully functional devices including integrated
electronics [108,109].
Conclusion
AM technologies have many potential biotechnological applications
ranging from the design of complex microfluidics to lab-scale devices
and direct fabrication of structures including living cells. The use of the
standardized .stl-format, which can be employed across different AM
technologies and printer devices, enables efficient open source dis-
tribution of innovative designs. Interdisciplinary projects involving
both engineers and scientists will be required to fully exploit the po-
tential of this innovative fabrication technology in bioprocess en-
gineering. Such projects could for example include CAD training ses-
sions for scientists and non-experts to educate them about the range of
possibilities and to encourage the spread of FabLabs and MakerSpace
institutions [110].
Acknowldegements
This work was supported by the German Research Foundation
[grant numbers STE 2232/3-1 and LO 1939/1-1] and the Saxon State
Ministry of Science and the Arts [grant number: 100239131].
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