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Integrated biopri

Department of Bioengineering, Stanford Uni

E-mail: ingmar@stanford.edu

† Electronic supplementary informa10.1039/c4ra05932h

Cite this: RSC Adv., 2014, 4, 34721

Received 18th June 2014Accepted 7th July 2014

DOI: 10.1039/c4ra05932h

www.rsc.org/advances

This journal is © The Royal Society of C

nting and imaging for scalable,networkable desktop experimentation†

Nathan D. Orloff, Cynthia Truong, Nathan Cira, Stephen Koo, Andrea Hamilton,Sean Choi, Victoria Wu and Ingmar H. Riedel-Kruse*

Adaptations of mass-market consumer electronics are increasingly used to aid experimentation in

engineering, life sciences, and education. Inspired by recent bioprinting and imaging research, we have

developed a low-cost, networkable, scalable bioprinter with integrated imaging that enables automated

fluid deposition with monitoring biological materials over a large area that can interface with standard

plasticware. We re-engineered a desktop printer and scanner with mechanical workarounds (rather than

software) to leverage the unmodified software, creating a complete system for �$700. We found that

the resulting print accuracy and precision of this system were close to those of the unmodified printer

and scanner. We demonstrate the reach of this system by printing multiple strains of Escherichia coli,

performing quantitative time-lapse recordings of bacterial growth, and then separating different

fluorescent strains according to color. A fluid-deposition and imaging platform, like the one developed

here, could be integrated for do-it-yourself research, remote experimentation, and (on-line) education.

I. Introduction

Repurposing inkjet printers and atbed scanners has led towidespread innovation across many elds, such as bio-fabrication,1–14 microuidics,15–20 materials science engi-neering,21 chemistry education,22 and biological imaging.27–29

Much of these bioprinting approaches have focused on tissueengineering applications that used an unmodied x-axis of theprinter (�200 mm) and length of the print-head (�20 mm).8,17,19

More recently, the components of a compact disk player andcustom soware14 were used. But in order to interface withconventional plastic-ware (e.g., multi-well plates, etc.), it isbenecial to signicantly increase the printing and imagingarea. By overcoming this challenge, we may enable future workon large-scale cell or organism migration assays,23,24 and time-dependent enzyme activity studies.26 Although bioprintingresearch has taken great strides, many efforts that use repur-posed inkjet printers require custom soware to expand theprint area.27–29 On the other hand, commercial bioprintersexist;11,25,26 however the cost, limited exibility of usablesubstrates, and print areas make scaling nancially prohibitivefor academic research.

Here, we report on an integrated platform that combines aconsumer level printer and a atbed scanner into a single unit(for the rst time) to enable repeated deposition of biological

versity, Stanford, 94305, California, USA.

tion (ESI) available. See DOI:

hemistry 2014

materials and imaging over a large-area (�700 cm2) at the sametime (Fig. 1 and ESI Movie†). Such a design closes the loopbetween the deposited materials and quantitative imagingfeedback required for controlled experiments with repurposedhardware. We recongured the printer hardware such as toprint over a stationary surface (rather than on a moving paper),and where we developed mechanical solutions to “trick” thesensors of the modied printer such that all the printer driversremained functional. Hence, the system does not requirespecial soware, making it amenable for scalable, networkabledesktop experimentation. The cost of this integrated bioprint-ing and imaging platform was approximately $700 (and couldbe decreased further).

In this paper, we rst provide a broad overview of the hard-ware architecture and the bypassed printer components neededto implement our approach. We then quantify the mechanicalperformance of the investigated bioprinter relative to theunmodied printer. Finally, we demonstrate that this systemcan be used to dispense “bio-inks” composed of live Escherichiacoli (E. coli), and then monitor the growth of these cells with theintegrated scanner. Throughout the paper, we will use the samenames and color-coding in the gures to identify the differentcomponents and states of the bioprinter. All of the image pro-cessing was carried out in MATLAB unless otherwise noted.

II. Bioprinter assembly

Our approach consists of three components: conguring thehardware to function as a atbed bioprinter, bypassing thevarious steps and checks in the state diagram with mechanical

RSC Adv., 2014, 4, 34721–34728 | 34721

Fig. 1 We developed a low-cost, large-area bioprinter with integratedimaging by mechanically re-engineering a commercial printer andflatbed scanner. (a) Top and side views of the assembled bioprinter andimaging platform. Four multi-well microplates demonstrate oneapplication (paper below is for contrast). (b) Corresponding schematicviews with a color-coded of the components. The same color-codingis used throughout. The modified shaft (*) and mechanical switch (**)are essential components, described in Fig. 3. (b) Staged photographsillustrating a single print cycle: “stalled in clutch”, “rev to print”, and“reset to switch” are states of reconfigured printer (see Fig. 2).

Fig. 2 We determined the state diagram of the unmodified printer andmodified the state diagram with mechanical components to enablebioprinter to use the commercial printer drivers. Diagrams of theunmodified state diagram (a) and the modified state diagram (b). Themodified state diagram removes two checks and their associatederrors from the state diagram. There are also two new actions (stalledin clutch, transition (trans) to drive) and a new step (reverse (rev) toprint). The modified state diagram enabled repeated printing over asingle print area.

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components, and integrating functional imagining. To reducedesign complexity and facilitate adoption by others,30 oursimplied design employs a commercial printer and scanner,and a handful of parts and fasteners (ve 3D acrylonitrilebutadiene styrene (ABS) printed parts, and three 2D laser-cutparts made from acrylic. See ESI† for more details). We chosethe HP DeskJet J1000-J110a printer, which uses HP 61 thermalink cartridges, and the HP G3110 atbed scanner (Fig. 1a). Aschematic of the bioprinter is shown in Fig. 1b. As in ref. 29, wealso used the SANE application programming interface tooperate the scanner. As the scanner did not require hardwaremodication beyond the plastic housing, we will focus on thesteps and components needed to modify the unmodied statediagram to “trick” the printer into repeatedly printing onto a 2Dstationary surface.

First, we disassembled the printer to understand theunmodied components and to identify components that couldbe modied to recongure printer architecture. In the process,we identied the optical slot encoder (a common designfeature) as the key component in our design, because it effec-tively acts like a switch, turning printing on and off. By

34722 | RSC Adv., 2014, 4, 34721–34728

understanding how the optical slot encoder functioned, we wereable to control the state diagram. For more details, see ESISection I.† From this, we designed a modied state diagram(Fig. 2b) that could be implemented by adding a custommechanical switch and modied paper feed sha with minimalchanges to the hardware and state diagram, preserving theprinter's ability to be controlled by conventional soware. Ourapproach essentially utilizes the motor that normally drives thepaper through the printer to advance the whole printer across a2D stationary surface; it also bypasses the calibration step,because it rotates the paper feed sha counterclockwise bymore than one page length. If we did not modify this statediagram, then the modied printer would not reset to the samey-position aer each print, and autonomous periodic printingonto the same 2D surface would not be possible.

To work around the calibration cycle in the printer rmware,we devised a duel rack-and-pinion system with a mechanicalclutch (Fig. 3a). The clutch consists of a spur gear mounted on aone-way bearing mated with a short clutch rack (Fig. 3b). Thesecond spur gear is directly connected to the sha, with a longer

This journal is © The Royal Society of Chemistry 2014

Fig. 3 We engineered a mechanical switch and modified the printershaft to enable repeated printing onto a stationary surface executingthe modified state diagram shown in Fig. 2b. (a) The assembledmodified paper feed shaft with acrylonitrile butadiene styrene (ABS)plastic parts (see Fig. 1b*). (b) Schematics of the modified shaft duringthe three action states of the modified state diagram (see Fig. 2b). Thecircular arrows in the side view show the rotation of the shaft in thedifferent states and the straight arrows show the resulting translation.(c) The ABS mechanical switch with the integrated paper feed slotsensor (see Fig. 1b**). (d) Schematics of the mechanical switch duringthe modified state diagram (see Fig. 2b).

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drive rack. This clutch is attached to the modied paper feedsha such that the printer only translates in the intendeddirections during the print cycle. The three panels in Fig. 3billustrate the states of the modied sha during the print cycle.

This journal is © The Royal Society of Chemistry 2014

The 12.13 mm-diameter modied sha consists of an ABSinsert, a 120 mm 4–40 threaded rod, two 4–40 nuts, a one-waybearing (Boca Bearing #HF0812), two brass 31-tooth 64-pitchspur gears (Calandra Racing Concepts #64031), a radial bearing(Boca Bearing #SMR6701-UU), and an ABS bearing sleeve. Thespur gear has a pitch diameter of 12.30 mm. The unmodiedrotary encoder wheel and timing wheel assemblies are reused topreserve print dimensions. Additional details of bioprinterassembly are provided in ESI Section II.†

Fig. 3c shows the mechanical switch with a ag that switchesthe paper feed optical sensor on and off. The slide with a agmoves along two 15 cm aluminum rails (3 mm in diameter) thatare secured in a 3D-printed custom housing (Fig. 3c). It isperhaps easiest to think of this component in the context of thestate diagram. When the printer is stalled in the clutch, the slotencoder is off (Fig. 3d). As the printer moves forward, a bumper(Fig. 1c) pushes the slide forward so that the ag blocks the slotencoder (Fig. 3d), switching the beginning of the print cycle. Atthe end of printing, another bumper pushes the slide back tothe initial position (Fig. 3d), which effectively bypasses the out-of-paper error to enable automated and repeated uid deposi-tion over a single area. Another slot encoder is covered with tapeto bypass the “output-tray-closed” error.

We completed the construction of our bioprinter by inte-grating a scanner. We found that the HP G3110 atbed scannerwas able to image objects that were up to 2 cm away from theglass with only minor distortion, which was desirable forimaging and printing on cell-culture plates. We modied theplastic housing of the scanner to maximize the overlap betweenthe scan and print areas. Other than modifying the plastichousing and threading holes for mounting, the scanner itselfwas not modied. We also modularly integrated the scanner sothat the bioprinter could function without the scanner, whichwas convenient for applications with a bench-top microscope.Fig. 1 depicts the completely assembled bioprinter with inte-grated imaging, which takes approximately two days tocongure based on our instructions (see ESI Section III†).

III. Print performance

Since we dramatically modied the hardware, it was necessaryto test the mechanical reliability of this bioprinter. During thesetests, we found that the printer was prone to gear-to-rackmisalignment when it translated quickly over distances greaterthan 1 cm and aer abruptly changing directions; motions thatoccur at the beginning and end of a print cycle and are a part ofthe rmware. Wemitigated this problem by printing a light-gray10 mm � 190 mm stripe at the top and bottom of the print areaand a thin gray border around the print area (Fig. 4a). The rack-and-pinion drive was robust, but it showed increasing wear overmany (�1000) uses and required occasional realignment. Whenerrors occasionally occurred due to alignment, the printerrequired amanual reset in order to clear thememory and restartprinting. Hence, the moving components (gears and racks) mayneed to be replaced over the lifetime of the bioprinter or couldbe constructed from sturdier materials.

RSC Adv., 2014, 4, 34721–34728 | 34723

Fig. 4 Print accuracy and precision of the modified printer are similarto those of the unmodified printer. (a) A 7� 8 grid containing a box-in-a-box pattern was used to quantify the accuracy and precision of theprinted image. (b) Data analysis procedure: top left, the binarizedimage of the box-in-a-box pattern. Top right, the correspondingedges images computed with a Sobel filter. Bottom, the sum of theedges along the y-axis as a function of the x-axis produced peakswhere the edges of the boxes could be easily defined (black, red, andblue denote outside, middle, and center boxes, respectively). (c) and(d) The resulting change in the position of the box along a given axisversus the intended position of the box. N represents the number ofbox lengths used to obtain each point and the associated error bars areone standard deviation.

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To characterize the accuracy and precision of the modiedprinter, we used the standard box-in-a-box alignment mark31,32

typically used in wafer fabrication. We drew a 7 � 8 grid of thebox-in-a-box pattern (Fig. 4a). The box sizes were 25.206 mm,12.603 mm, and 1.401 mm with line widths of 0.706 mm, 0.353mm, and 0.176 mm, respectively. We printed the box-in-a-boximage on ve separate sheets of paper and scanned the imagewith a atbed scanner at 600 dpi.

We analyzed these printed box-in-a-box patterns by con-verting the image to grayscale, cropping each box-in-a-boxpattern to the same position, binarizing it, and summing it inthe x- and y-directions separately to obtain distinct peaks(summing in the y-direction is shown in Fig. 4b). From thesepeaks, we obtained three independent measurements: thewidth of the box and both line widths at the far and near edges.This procedure was carried out for every box along each axis. Forthe 1.401 mm box, we only computed the width of the box tosimplify the analysis. We also obtained the centroid of each box,which was ultimately sufficient to characterize the printperformance. The orientation of the y-axis of the page relative tothe printer and scanner was within 0.1� and we veried thealignment by tracking the edge of the page for the ve prints.

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More details are provided in the ESI Section IV along with theanalysis code (Supplemental Materials, bibanalysis.m).†

Following the standard box-in-a-box approach,31,32 we wereable to characterize the behavior of the modied bioprinter.Fig. 4c and d shows the difference between the intended posi-tion of the box and the actual position of the box as a function ofintended position for each axis. A linear trend as a function ofthe y-position is observed, which resulted from a mismatch ofthe spur gear pitch diameter (12.30 mm) relative to the diameterof the paper feed sha (12.13 mm). The cumulative offset was2.8 mm for a 297 mm-long page. The average accuracy of themodied printer was 0.22 mm � 0.02 mm (x-position) and 1.21mm � 0.10 mm (y-position), which was comparable to those ofthe unmodied printer. Note that the accuracy values are ofprimary relevance when it comes to repeated printing over thesame area, but less so for a single print.

IV. Applications in bioprinting

Various bioprinting applications with modied ink-jet printershave been demonstrated in the literature, many of which canalso be performed with our platform.1–14 Since the currentinvestigation focused on the hardware aspects of the bioprinter,we only demonstrate three examples of applications that werespecically enabled by the larger print area and simultaneousimaging capability of this device. These example applicationsused various E. coli strains (TOP10) that had been transformedwith plasmids expressing the uorescent proteins Cerulean,mCherry, and Venus.33 Live cells were incubated overnight at 37�C in lysogeny broth (LB) supplemented with chloramphenicolto select for cells carrying the vectors with the uorescentproteins. The cell suspension was then loaded directly intocleaned ink cartridges.12 We were able to use both three-color(deposition of multiple strains) and black ink cartridges(deposition of a single strain). We found that we were able toprint many water-based liquids without modication. In somecases, we also found that sucrose could be used to tune theviscosity and improve the print performance.26

For simultaneous printing of multiple strains, we loaded theuorescent strains into the cyan (Cerulean), magenta(mCherry), and yellow (Venus) reservoirs of a three-colorcartridge. Once this “bio-ink” was loaded into the cartridge, weprinted various patterns of cells onto chloramphenicol-supple-mented LB agar cast in Petri dishes (Fig. 5a, le). In thisexperiment, we inverted the image to achieve the correct colorsin the printed composite. The printed plate was incubated at 37�C for six hours to promote E. coli growth and then imaged witha conventional uorescence microscope (Leica M205FA,DFC310FX, LAS AF). To create these images, we used a pano-ramic stitching program (Microso Image Composite Editor) tojoin 40 images; the total image size was limited by the uores-cence microscope. Once the panoramic stitching programassembled the Cerulean, mCherry, and Venus channels, werecombined the images to create the composite image by con-verting the channels to grayscale, tuning the contrast of eachimage separately to account for saturation. We then assignedthe images to the cyan, magenta, and yellow channels to create a

This journal is © The Royal Society of Chemistry 2014

Fig. 5 The modified printer simultaneously deposits multiple fluo-rescently labeled E. coli strains onto an agar surface utilizing a three-color ink cartridge. (a) Bioprinter configured for two 150 mm Petridishes with Physarum polycephalum cultures. A white paper sheet wasadded for contrast. (b) Left panels show the individual cyan, magenta,and yellow channels imaged over the same area. Each channelconsists of an E. coli strain that had been transformed with custom-designed vectors to express a single fluorescent protein. Thecomposite image is the inverted combination of each channel. Otherthan modifying the intensity of each channel for combination incomposite, the images are unaltered.

Fig. 6 The bioprinter-imaging platform enables quantitativemeasurement of bacterial growth and color-based separation of flu-orescently labeled E. coli. (a) Growth curves for diluted cell suspen-sions were obtained via grayscale values acquired with the scanner atroom temperature. The solid lines are fits to the data with the Baranyimodel. Selected images from the scanner at 25 h, 35 h, and 45 h for the100% grayscale print are shown. For these images, we modified thecontrast for better visibility. Error bars: standard error of the mean forfive replicates. (b) Truecolor image from the scanner after backgroundsubtraction, and the same image in the mCherry (m), Venus (V), andCerulean (C) colormap spaces. WT, wild-type. (c) Three-color plotsused to transform the RGB colormap into mCherry, Venus, andCerulean colormaps. The gray circles are the background, which weset to zero.

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single image, which was inverted to obtain the composite image(Fig. 5b, right). We noticed that the image from the cyanchannel had large streaks, likely due to partially clogged ordamaged nozzles.9 Despite this imperfection, images ofcomparable print quality were typical for this bioprinter andwere very accurate over a distance in comparison to theliterature.6–14

Next, we assessed whether the imaging component of thesystem was capable of capturing quantitative bacterial growthdata over time. In these experiments, we loaded the blackcartridge with wild-type E. coli (i.e., a lab strain that did not

This journal is © The Royal Society of Chemistry 2014

express a uorescent protein) and removed the color cartridgefrom the printer. We printed four grayscale values, nominally100%, 50%, 25%, and 12.5%, demonstrating that we were able totune the initial number of cells in the bacterial colony depositedon the plate. Each printed colony had an initial diameter of 5mm. The colonies were imaged with the atbed scanner every 30min for a total of 60 hours at room temperature to quantify thegrowth of wild-type E. coli (Fig. 6a). To understand the growth ofthe E. coli, we performed a few image processing steps (see ESISection V†) to obtain the colony area fraction. Aer repeating theexperiment ve times, we obtained the mean and standarddeviation of the colony area fraction, which we normalized bythe mean colony area fraction of the 100% culture at 60 h.

To quantitatively compare our growth curves to other resultsin the literature, we t each curve independently to the Baranyimodel (solid lines in Fig. 6a).34 The Baranyi model is given as,

RSC Adv., 2014, 4, 34721–34728 | 34725

Table 1 Fit parameters for Baranyi model

Dilution yf [au] mmax [1/h] l [h] yo [au]

100% 8.60 0.15 5.00 2.8350% 8.13 0.16 12.50 2.5725% 7.85 0.17 12.50 1.6812.5% 7.39 0.15 22.80 3.04

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yðtÞ ¼ yf þ ln

� �1þ emmaxl þ emmaxt

�1þ emmaxt þ eðmmaxl�yf�yoÞ

�(1)

where mmax is related to the doubling time, l is related to the lagtime, yf is the natural logarithm of the nal area, and yo is thenatural logarithm of the initial area.34 From eqn (1), we obtainedcolony area doubling times (tarea¼ ln(mmax)/2) ranging from 4.06h to 4.76 h. Since the conventional population doubling time isproportional to the volume of a colony, we expected our areadoubling time to be related by tvol ¼ tarea

2/3. This calculationyielded doubling times from 38 min to 43 min for our cultures,which was reasonable given that these experiments were per-formed at room temperature and growth may have occurred inthree dimensions. The remaining t parameters are shown inTable 1.

Next, we sought to demonstrate that the scanner could beused to distinguish uorescent strains based on the visiblecolor measured by the scanner. Given the ubiquitous usage ofuorescent probes, this feature would enable a much widerrange of applications in the life sciences. To demonstrate thisability in an unmodied scanner, we manually plated theCerulean, Venus, mCherry, and wild-type strains individuallyonto chloramphenicol-supplemented LB agar plates, incu-bating the plates at 37 �C for six hours, and then imaged thecolonies with the scanner (Fig. 6b). The resulting imagecontains information in the standard red-green-blue (RGB)channels. We computed a linear regression from themean colorvalue to the origin of the plot for each of the strains in this RGBspace. As an example, the red-green sub-space is shown inFig. 6c. These regression lines were then used to create a colormap transformation from the RGB space into the “mCherry,Venus, Cerulean” color space (Fig. 6b). This approach enabledus to use the scanner to differentiate between the mCherry- andVenus-labeled strains; however, the Cerulean-labeled strain wasnot easily distinguished from the wild-type. Higher expressionlevels or adapting the scanner for uorescence imaging wouldyield additional color-separation capabilities.

V. Conclusions

Here we have re-engineered a commercial inkjet printer andatbed scanner into an integrated uid-deposition imagingplatform by leveraging much of the unmodied architecture ofhardware and soware, rather than developing new soware orcustom microcontrollers. This design approach is thereforeamenable for integration within a standard desktop andnetwork environments, and the performance (speed, accuracy,

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precision) is close to that of the unmodied hardware. Some ofthe shortcomings that we discussed, which are intrinsic toreverse engineering, could be overcome in a commercial mass-market product. For example, adding optimized lters couldenable uorescent imaging and the calibration cycle could beomitted in the printer rmware. Although commercial instru-mentation will not likely use our mechanical solutions, thiswork demonstrates that the existing infrastructure can beadapted to facilitate life-sciences experimentation over anetwork without extensive programming or custom soware.

The presented platform can print a wide variety of “bio-inks”beyond the E. coli inks tested here, such as sugar water, saltwater, and many of the nonconventional inks demonstrated inprevious reports.4 To showcase these new capabilities, we usedthis bioprinter to create complex cell depositions over a largearea with accuracies that were comparable to those of anunmodied printer. Furthermore, the distance between printhead and the support surface (i.e., scanner) can be modiedwith rubber lis to easily accommodate different substrates,materials, and plastic-ware. By integrating a scanner, we addedanother dimension to consumer-based bioprinting research,enabling real-time monitoring of deposited cells and uids orautomated feedback. Such capabilities could enable the depo-sition of complex chemical gradients, experiments to study therelationships between different cell cultures, studies onquorum sensing, and other applications including drugdiscovery.1–14,18

In summary, the large-area bioprinter with integratedimaging presented here could facilitate bioengineeringresearch, and even empower science education, both locally andon-line.35–38

Acknowledgements

The BioX IIP Grant at Stanford University and the StanfordDean's Postdoctoral Fellowship Program supported thisresearch. Component fabrication was performed at the ProductRealization Laboratory and the Department of Bioengineeringat Stanford University. NDO and IHRK conceived and designedthe hardware, and the experiments. CT and SK assisted with theexperiments, hardware assembly, and design. NC, VW, and AHhelped perform the experiments with E. coli. SC assisted withdata analysis. We thank Peter Redl, Alice M. Chung, ZahidHossain, David Glass, and Paulo Blikstein for their insightfuldiscussions during the course of this research.

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