Annals of Biomedical Engineering (Ó 2015) DOI: 10.1007/s10439-015-1289-4

Design and Fabrication of an MRI-Compatible, Autonomous Incubation System VAHID KHALILZAD-SHARGHI1 and HUIHUI XU2 1

Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA; and 2School of Engineering and Computer Science, University of the Pacific, 203 Anderson Hall, Stockton, CA 95211, USA (Received 13 November 2014; accepted 23 February 2015) Associate Editor Xiaoxiang Zheng oversaw the review of this article.

Abstract—Tissue engineers have long sought access to an autonomous, imaging-compatible tissue incubation system that, with minimum operator handling, can provide real-time visualization and quantification of cells, tissue constructs, and organs. This type of screening system, capable of operating noninvasively to validate tissue, can overcome current limitations like temperature shock, unsustainable cellular environments, sample contamination, and handling/ stress. However, this type of system has been a major challenge, until now. Here, we describe the design, fabrication, and characterization of an innovative, autonomous incubation system that is compatible with a 9.4 T magnetic resonance imaging (MRI) scanner. Termed the e-incubator (patent pending; application number: 13/953,984), this microcontroller-based system is integrated into an MRI scanner and noninvasively screens cells and tissue cultures in an environment where temperature, pH, and media/gas handling are regulated. The 4-week study discussed herein details the continuous operation of the e-incubator for a tissueengineered osteogenic construct, validated by LIVE/DEADÒ cell assays and histology. The evolving MR quantitative parameters of the osteogenic construct were used as biomarkers for bone tissue engineering and to further validate the quality of the product noninvasively before harvesting. Importantly, the e-incubator reliably facilitates culturing cells and tissue constructs to create engineered tissues and/or investigate disease therapies. Keywords—MRI-compatible, Magnetic resonance imaging, Bone tissue engineering microcontroller.

INTRODUCTION There is a great need for the development and evaluation of an incubation system that not only uses medical

Address correspondence to Huihui Xu, School of Engineering and Computer Science, University of the Pacific, 203 Anderson Hall, Stockton, CA 95211, USA. Electronic mail: [email protected], hxu@pacific.edu

imaging technologies, but also closely simulates in vivo conditions and enables observation of cell and tissue constructs.1,19 As compared to conventional tissue culture systems, an autonomous in vitro culture-imaging platform allows for continuous monitoring and recording of the culture environment. In addition to assisting with the ex vivo investigation of the pathophysiology of diseases, such platforms have potential applications in regenerative medicine, drug delivery studies, as well as basic biological research.16 However, a limited number of tools exist that can noninvasively extract information from the cultured constructs without causing destructive changes to their structure. Imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) are considered to be among the best validation methods. In particular, MRI scanning is a noninvasive technique that uses non-ionizing radiation. Moreover, MRI is a leading tomographic method for observing the growth and metabolic functions of cultured cells and constructs.22 Likewise, for tissue-like constructs, MRI can help determine the distribution of cells inoculated, the global density, flow dynamics, and quality control in cell distribution.8 MRI also provides superb image quality, high spatial resolution, varied contrast-weighting, 3Dmultiplanar capability.5,6,15 Specifically, Petersen et al. developed an MR-compatible hollow fiber bioreactor (HFBR) capable of growing cartilage tissue in the bioreactor’s extra-capillary space of the bioreactor.13 Chesnick et al. applied another HFBR bioreactor to quantify bone tissue growth using MRI.3 However, while significant advancements, both studies failed to accurately measure the parameters of the culture environment. Also, susceptibility artifacts due the hollow fiber compromised the MR images.

Ó 2015 Biomedical Engineering Society

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Within the field of tissue imaging, it is critical that the repeated imaging of a tissue construct be performed in a noninvasive manner and does not harm the developing tissue. However, most current systems cannot offer a completely noninvasive means for characterizing the culture. Therefore, the samples risk of exposure to contamination, temperature shock, or handling difficulties from the incubation platform where they are being assessed.11,23 This highlights the need for an autonomous incubation system that can function in tandem with an imaging machine and allow for repeated visualization of variations in the structure and characteristics of the culture. In the current study, our novel, small-scale, portable, and autonomous system, known as the e-incubator was applied to visualize cultured tissue constructs and biological compounds of miniscule sizes and different geometries in real-time and in a closed controlled environment. The design addressed the need for a system with realtime tomographic imaging capability, which is provided for by integrating the imaging platform with a high-field MRI system. Further, all applied aspects of the study were designed and adapted to facilitate MR imaging and reduce MR signal loss. The e-incubator design offers a sterile environment, control over culture conditions (e.g., temperature, CO2 level, nutrient supply, waste drain, and growth factors), transparency to MR signal, accessibility of the culture for imaging, and media/gas reservoirs. Additionally, the e-incubator’s chambers (including necessary components) are designed to be nonmagnetic and compact enough to readily fit inside the relatively narrow bore of the most MRI micro-imaging systems. Further, the e-incubator places the culture in the center of the field-of-view (FOV) while other aspects of the instrument can be outside of the scanner. Also, the eincubator can be easily inserted in and out of the MRI scanner. In a preliminary investigation various materials, electronics, sensors, mechanics, connections, biological materials and associated strategies were under examination to conclude the final design. Mesenchymal stem cells seeded into a gelatin scaffold sponge were used to create a construct capable of producing bone tissue. Through this examination, we were able to demonstrate the functionality of the system for the osteogenic construct within a 4-week study. We used NMR parameters as quantitative biomarkers for bone tissues; by continuously imaging the culture with the MRI system, we investigated the 3D structural changes of the construct during its growth. A bone tissue engineering study recently validated the e-incubator.12 The purpose of this paper is to describe the design of the e-incubator system and demonstrate its functionality.

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MATERIALS AND METHODS System Description For the e-incubator, all components inserted inside the MRI must be MRI compatible. These include the chambers, sensors, culture well, holder, and the connections between the components. Figure 1a represents the block diagram of the e-incubator system including components and connections within it. The system contains two chambers (Fig. 1d) attached to a specially designed holder. The top chamber houses the culture, and the bottom chamber serves as a miniature water bath for heating purposes. Both chambers and the holder are machined from polycarbonate, which is biocompatible, autoclavable, MR-compatible, and sturdy across a large temperature range. The culture well, shown in Fig. 1e, is designed to hold the construct. The Dulbecco’s Modified Eagle Medium (DMEM), being pumped into the culture chamber, can generate air bubbles that affect the homogeneity of the magnetic field. To avoid this issue, the culture well was tailored to filter the bubbles and conduct them away from the construct. The elements are integrated such that fresh media is supplied to the construct in the culture chamber. The culture mini-environment, including pH, temperature, and gas mixture level, is monitored and controlled. Data are recorded online and transferred to a personal computer (PC). The construct is imaged in real-time. Due to the limited space available and the high magnetic field used in this study, elements must be carefully selected to fit the maximum diameter of 35 mm and tested to ensure imaging transparency. It has been shown that the diameter of the culture chamber must be a few millimeters smaller than the radio-frequency (RF) coil to reduce the inductive and capacitive coupling.8 To take this into account as well as to allocate the space required to accommodate the bubbling of the gas, the diameters of the chambers were reduced to 32 mm; this diameter also readily fits inside the 40-mm RF coil. To optimize the sensitivity of the RF coil, the heaters, the temperature probe and the overheating circuit were mounted out of the imaging field-of-view (FOV). Furthermore, the wiring for the temperature sensor was conducted though the top of the magnet so as not to pass through the FOV. The culture environment including temperature, pH, and CO2 level has a significant influence on the quality of the cultured product.7,10 In the course of normal growth, the pH level of media decreases due to the cell metabolism and degradation of the applied scaffold. At 1-min intervals, the media pH is measured using a pH sensor (Atlas Scientific, Brooklyn, NY). This sensor was mounted in the middle of the media

Medical Imaging-Compatible Autonomous Incubation System Gas Inlet

CO2/Air Mixing System

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Heater

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RF Field

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Scanner (c)

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Heating Chamber Culture Chamber

Gradients Coils

Peristaltic Pump

Cross-Section View

(a)

Chambers (d)

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FIGURE 1. Schematic overview of the e-incubator. (a) The Microchip microcontroller supervises the accurate operation of the whole system. (b) The chambers are attached to an imaging holder and placed in the center of the MR scanner. (c) Vertical bore MRI magnet used to real-time monitor the culture. (d) Cross sectional view of the exploded assembly of the chambers: Top chamber is the culture chamber and bottom chamber is the heating chamber in which a tube is mounted to direct the media through to the culture chamber. A silicon rubber heater is wrapped around the tube. (e) 3D view of the culture well especially designed to house the culture and avoid generating bubbles into the field of view. It includes a well with a cone-shaped base with one-half millimeter holes in it.

line from the culture chamber to the media reservoir. Also, the sensor was a full-range pH sensor reading from 0.01 to 14.00; this range provides scientific grade readings with offset of ±0.20 pH. The corresponding recorded data were used to calibrate the media exchange intervals. To maintain pH stability (7.0–7.5), 5% CO2 gas exchange took place inside the culture chamber every eight minutes. To preserve the temperature of the culture media at 37 °C, an MR-compatible thermal probe with accuracy of 0.1 °C, and an air blowing heating system (SA Instrument, Inc, Stony

Brook, NY) along with a silicone rubber heater (O.E.M. Heaters, Saint Paul, MN) were employed. The silicon rubber heater was mounted inside the heating chamber and a flexible air duct, connected to the blowing heater, was inserted from the top of the magnet’s bore to direct the warm air toward the culture chamber. Notably, the temperature probe inside the media reports temperature readings back to the heaters; these readings caused both heaters to toggle on and off as needed, leading to the desired temperature.

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MFC

Outlet

To maintain the concentration of nutrients and metabolites, as well as to move the waste away from the cells, the media inside the culture chamber must be replaced with the fresh media.10 For this purpose, a peristaltic pump (Thermofisher Scientific, Barrington, IL) with an adjustable flow rate, here 6 ml/min, was used to circulate the media between the culture chamber and the media reservoir, which was refilled weekly with 100 ml of fresh media. Media inside the reservoir was kept at 38 °C because it loses nearly 3 °C while pumping through the tubing. During pumping, heaters were turned on automatically to increase the temperature to 37 °C in about five minutes. Another benefit of the periodic media exchange is that it backs up the media level inside the culture chambers at the default level compensating for evaporation. All fluidic connections were made using 2-mm-bore tubing and luer lock connectors; leakage was precluded by means of O-rings. In order to equilibrate the media in the desired pH range, a gas mixing system was designed to produce a gas mixture containing 95% air and 5% CO2. This system utilizes two mass flow controllers (MFCs) (Cole-Parmer, Vernon Hills, IL) that are calibrated to work as either air or CO2 flow meters/controllers. The MFCs operate in an extensive range of flow and with accuracy of ±2% full-scale (at 20 °C and 30 PSIG). One of MFCs was connected to the laboratory compressed air line regulated to the pressure of 10 psi, and the other MFC was connected to a CO2 tank regulated to 8 psi, as shown in Fig. 2. The outlets of both MFCs were connected to a T-junction by 5-mm-bore PVC air tubing, and the mixed gas line was connected to the gas

5% CO2

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FIGURE 2. Diagram of the designed gas mixing system. Two mass flow controllers were used to mix 5% CO2 with 95% air. Three Whatman Hepa-Vent Filters (GE Healthcare Life Sciences, Piscataway, NJ) were employed to ensure clean airflow.

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inlet of the culture chamber. The flow of each individual MFC was regulated by the system controller and required initial calibration according to the back pressure to which it was exposed. Because the gas flow, not the pressure, is regulated by the MFC, the back pressure to which CO2 gas line is exposed in the Tjunction and higher air pressure are taken into account in order to readily achieve the desired CO-2 level in the gas mixture. The 5% CO2 is maintained by turning on the MFCs for Ta time and switching it off for the longer Tb time. For this small-scale system, Ta and Tb times were 30 s and 8 min respectively. The CO2 concentration was constantly monitored using a CO2 sensor (CO2 Meter, Ormond Beach, FL). The gas flow shuts down during the media exchanged in order to prevent foam from forming on top the surface of the media; in turn, the gas can exit through the gas outlet line. Likewise, outgoing gas with relatively high humidity flows from the culture chamber toward the CO2 sensor, causing the water vapor to condense into the liquid water and accumulate before the air filters. The resultant wet air filter is unable to ensure the sterility of the system and can lead to contamination. Additionally, the calibration of the CO2 sensor is altered due to the very high level of humidity. To reduce these risks, a 1-L air flask was installed between the outlet of the culture chamber and the air filter. The gas pressure has the tissue construct exposed to the intermittent hydrostatic pressure through the culture media. This gas pressure is transferred to cells through the media, which was previously shown to have influence on the growth of cell culture particularly for the articular cartilage.9 The sequence and duration of loading are converted into cell signaling, which moderates the metabolism of the cells. Control System Microcontrollers and programmable logic controllers (PLCs) have been used to automate measurement collection and the control of the events, as well as to collect/process the data. In order to efficiently operate the e-incubator, a new control module was designed based on the control requirements of the platform, and a related circuit was mounted on a single-side raster board. The interactions between the microcontroller and the other components of the module are illustrated in Fig. 3. For the control module of this system, a PIC16f917 microcontroller (Microchip Technology Inc., Chandler, AZ) was selected because of the wide range of features it provides. PIC16f917 is an 8 bit model with 14 k Byte programmable flash memory. It includes 8 channels inbuilt 10-bit resolution analog-to-digital convertors and supports standard serial communication, which can

Medical Imaging-Compatible Autonomous Incubation System Temperature Sensor

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PIC 16f917 Microcontroller

Peristaltic Pump

Heaters

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FIGURE 3. The interactions between the microcontroller and the other components of the control module. The Microchip microcontroller receives inputs from the sensors and based on the programed algorithm makes decisions to control the system. All the data were sent to a PC and recorded by a windows based interface.

reduce the complexity of the algorithm needed for the operation of the system. Communication with the pH sensor was performed using the standard serial protocol (RS232). For this purpose, a universal synchronous asynchronous receiver transmitter (USART) module of the microcontroller was used in asynchronous mode. To exchange data with a PC, a software-based serial method compatible with RS232 was used because the PIC16f917 has only one USART. A MAX232 integrated circuit (Texas Instruments, Dallas, TX) was employed to convert the signal from unipolar 5-volt TTL/CMOS levels of the microcontroller into the bipolar standard signals TIA/EIA-232-F (a standard PC COM-port) that are required by PC. The analog output of the temperature sensor was amplified and connected to the first analog to digital converter channel of the microcontroller and converted to digital signal. To eliminate temperature fluctuation caused from the noise, the analog values were measured 10 times over 500 ms and averaged to reach to a smooth change. The microcontroller software was developed in C high-level programming language using Microchip MPLAB Integrated Development Environment (IDE) and compiled by Microchip HI-TECH C Compiler for PIC10/12/16. PICKit2 programmer wrote to the program memory of the microcontroller. The circuit of the module was designed using the Altium designer software (Altium Ltd., Sydney, Australia). The microcontroller software is primarily composed of an initialization routine, a main control routine, and a brief service interruption routine. While operating, the main control program is interrupted by the execution of the service interruption routine every 100 ms; this service interruption routine is triggered by overflow of the timer 1 available on-chip. The service interruption routine includes program steps for keyboard reading, along with a control routine for the CO2 injection system. Figure 4 describes the flowchart

Main program gate

CO2 interval =8 min

Initialize variables, registers, and ports

No

Yes Initialize LCD, pump and CO2 system

Turn on injection for 20 second, Update LCD and heaters status every sec, Send data to PC

Run startup function

Read temperature (T) and pH

T37 Yes Heaters off

Exchange media Update LCD and Heaters status every sec, Send data to PC

FIGURE 4. The flowchart of the main control routine.

of the software, including the main control routine. The software was stored in the memory of the microcontroller. When the system turns on, a startup function runs after setting all necessary initializations in order to pump out the media to the media reservoir and pump the fresh media in; therefore, in the case of temporary power outage, media in culture chamber returns to its normal level. During the shutdown stage, the heaters and CO2 system turn off, and the media returns to its

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reservoir. Also, the control system includes a circuit to protect against overheating. This circuit causes the heaters to shut down in the event that the temperature probe fails. To do so, the software calls a routine specifically designed to read the temperature and pH, compute the calibrated values, show on a LCD, and send this information to a PC using an RS232 serial protocol. Data recording is an essential requirement of biological systems for computer aid processing and validation of products. All data collected by the control module was transferred to a PC and recorded by a software interface. Additionally, a surveillance camera (D-Link, Taipei, Taiwan) was used for online observation of the real-time status of the system. Magnetic Resonance Microimaging System Subsequent to loading the sample into the culture chamber, the chamber’s assembly was attached to a specially-designed holder (Fig. 1b) and placed into a 9.4 T (400 MHz for protons) 89 mm vertical-bore MRI scanner (Agilent, Santa Clara, CA) as shown in Fig. 1c. The scanner was equipped with triple-axis gradients with maximum strength of 100 G/cm. A 4 cm Millipede RF coil RF performed excitation and signal reception. The imaging software VnmrJ 3.1 performed the power and frequency calibration, imaging plane alignment, and parameters entrance. Imaging sessions were scheduled between media exchanges, which take approximately every 8 min. Notably, the system allows for image acquisitions through remote desktop applications such as VNC Viewer (RealVNC Ltd, Cambridge, UK) or smartphone apps; thus, the operator after initial setup can run the system remotely. Validation Experiments Given that the designed system is inserted into the MRI scanner, it is important to investigate how the system influences the homogeneity of the related magnetic field. Therefore, this influence should be investigated prior to the cultivation experiment. MRI phase difference maps contain information about the homogeneity of the magnetic field and can be utilized to compute phase difference maps for inhomogeneity corrections to the magnetic field (i.e., the source of contrast in phase images equals the differences in the precession frequencies (Dx), which depend on the local homogeneity of the magnetic field). It has been shown that areas with high topography of phase difference maps can be a sign of inhomogeneity artifacts, which are exacerbated with longer echo time (TE).14,21 To generate phase difference maps, first two gradient-echo images were acquired with different echo times (TEs)

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and then phase difference (Du) was calculated by subtracting the phase images. In order to correct the probable phase wrapping in the phase difference map, several algorithms have been developed to map the phase in the range of 2p to p.17 Thus, without installing the temperature sensor, and with the heaters turned off, the phase difference maps were generated for the culture chamber filled with DMEM. The associated phase difference maps were generated for TE1 = 2.56 ms (minimum), TE2 = 10 ms acquired by standard gradient-echo scans with a repetition time (TR) of 300 ms, flip angle of 20, matrix size of 128 9 128, a slice thickness of 1 mm, NEX = 1, and a FOV of 23 mm2. After installing the temperature probe and turning the heaters on, the MR acquisitions were performed again using the same parameters, and phase difference maps were created. Additionally, the full-width half-maximum (FWHM) spectral line width, which is used as an index of the magnetic field homogeneity, was also measured before and after installing the temperature sensor and turning on the heaters. A 4-week study was designed in order to evaluate the performance of the system for both maintenance of the culture conditions as well as MR imaging compatibility. Two Gelfoam sponges (Pharmacia & Upjohn Co., Kalamazoo, MI) were seeded with human mesenchymal stem cells (hMSCs) purchased from Lonza Walkersville, Inc., at 1 9 106 cells/ml and treated with osteogenic media as previously described.12 After 48 h of static culture, one of the constructs was placed in the e-incubator to differentiate the osteogenetic lineage. Additionally, the other construct was incubated in a standard incubator (Moriguchi, Osaka prefecture, Japan) under the same culture conditions. During the course of the 4-week study, axial fast-spin-echo MRI images were acquired daily (repetition time TR, 2000 ms; echo spacing ESP, 20 ms; echo train length ETL, 4; number of averaged experiments NEX, 16; field of view FOV, 23 9 23 mm; matrix, 256 9 256 pixels; slice thickness, 1 mm). To measure the alteration of T2-relaxation time axial, multiple-echo-spin-echo MRI acquisitions (TR, 4000 ms; effective echo time TE, 10 ms; number of echoes NE, 64; NEX, 2; FOV, 23 9 23 mm; matrix, 128 9 128 pixels; slice thickness, 1 mm) were performed. The acquisition of a series of spin-echo-multislice-diffusion-weighted (SEMSDW) images generated the apparent diffusion coefficient (ADC) data (TR = 1000 ms; TE = 27.04 ms; FOV = 23 9 23 mm; NEX = 2; slice thickness = 1 mm; using a 128 9 128 matrix). The diffusion gradient was applied in the readout direction with a b value of 1200 s/mm2, diffusion gradient duration (d) of 3 ms, and a separation (D) of 18 ms. MRI images were acquired with a

Medical Imaging-Compatible Autonomous Incubation System

transaxial resolution of 90 lm. Obviously, MRI acquisitions must be scheduled between media exchanges. Also, a matrix laboratory (MATLAB)-based program was used to calculate the T2 and ADC data for image processing. After image acquisitions, data were transferred to a PC and processed in steps such as masking, noise reduction, and curve fitting. All measures were presented as mean ± standard deviations of the values of all the pixels within the region of interest (ROI) which were calculated within the construct. On the 28th day of the study, both the constructs cultured in the e-incubator and the standard incubator were harvested and cut in half for Live/Dead assay and histology studies. Live/Dead assays were carried out using calcein AM/Ethidium homodimer fluorophores to discriminate between live and dead cells, which are identified by green and red fluorescence respectively. In addition, von Kossa staining was employed asses constructs and confirm if osteogenic differentiation was successful. For additional quality control measures, all slides were imaged with a light microscope AX70 (Olympus, Center Valley, PA) and examined by a certified pathologist.

RESULTS Validation of the e-Incubator System After instrumentation development, validation tests were used to evaluate the performance of the system. Figure 5 shows the temperature, CO2, and pH data recorded by the software interface in 24 h period. The average and standard deviation of the temperature and

Results for Bone Tissue Engineering Figure 7 denotes changes in the magnitude fastspin-echo images of the e-incubator-cultured construct during the 4-week study. Axial images for the same slices were taken daily during the study. Image analysis showed that the side dimension of the construct reduced in from 5.8 to 4.7 mm, and the thickness reduced from 2.8 to 2.2 mm. Due to the calcification, the reduction in the MR signal intensity increased as the study progresses, leading to darker images compared to the onset of the study. Figure 8a shows the average T2-relaxation times of the construct measured daily; these times depict decay from 143.4 ± 6.1 to 64.1 ± 6.4 ms. Additionally, Fig. 8b shows the T2 maps generated for four time points. ADC values were measured daily, and ADC maps were calculated weekly, as illustrated in Figs. 8c and 8d, respectively. The average ADC for the ROI

π

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pH

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Temperature, CO2, and pH Data

CO2 percentage were calculated as 37 ± 0.4 °C and 5 ± 0.3% respectively. Because the pH probe was installed in the middle of the media line from the culture chamber to the media reservoir, the recorded pH dropped from 8.4 to 7.8 while the media was pumped out of the culture chamber. Figure 6 illustrates that the magnetic field homogeneity were not altered for the culture chamber filled by DMEM media before and after installing the temperature probe and turning the heaters on. No significant differences were observed on the magnetic field map (Hz) after installing the temperature sensor and turning on the heaters. In addition to the phase difference maps, the 1H spectroscopy acquisitions performed on the culture chamber including the culture well represent that the corresponding FWHM increases from 93.5 to 103 Hz respectively (from 0.23 to 0.26 ppm).

CO2 pH Temperature

Time (Hours)

FIGURE 5. Temperature and pH contents in the media as well as CO2 concentration inside the culture chamber during 24 h. During the media exchange, which occurs every 6 hours, temperature and CO2 values are not reliable and show up as narrow valleys on the graph. The pH values are reliable when the media inside the culture chamber reaches the pH probe while being pumped out towards the media reservoir.

0

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(a) Magnitude Image

(b)

(c)

FIGURE 6. Influence of the heaters setup and temperature sensor on the homogeneity of the magnetic field. a Axial magnitude gradient-echo MRI image of the culture chamber filled with DMEM. Imaging parameters are: TR 5 300 ms, TE 5 2.56 ms, flip angle 5 20, Matrix 5 128 3 128, Slice Thickness 5 1 mm, NEX 5 1, FOV 5 23 mm2. b Phase difference map (describing magnetic field homogeneity acquired of the culture chamber without the temperature sensor and with heaters off. c phase difference map acquired of the culture chamber with the temperature sensor and with heaters on.

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Live/Dead assay

Incubator osteogenic construct

50 μ

50 μ

Histology

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FIGURE 7. Axial magnitude fast-spin-echo images of the cultured construct during the cultivation of a Gelfoam sponge seeded by hMSCs performed to validate the function of the system. The images were acquired daily but only one image for each week is illustrated. The side dimensions of the construct are shown on the images. The dimension is the approximate thickness of the construct assuming a homogenous slice.

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FIGURE 9. (a) Live/Dead assay for the construct cultured inside the incubator. (b) Live/Dead assay for the construct cultured inside the e-incubator. Live cells and dead cells are represented with green color and red color respectively. Three random live cells and dead cells were denoted with arrows and dashed circles correspondingly. (c) von Kossa staining for the incubator–cultured construct. (d) von Kossa staining for the e-incubator–cultured construct. The cells are stained pinkish-red and mineralized areas are stained black. von Kossa staining demonstrates calcium deposits in both specimens.

values decreased from 1.8 ± 0.1 to 1.5 ± 0.2 mm2/ s 9 1023. At the conclusion of the study the two constructs (i.e., incubator-cultured and e-incubator-cultured) were harvested and cut in half. The subsequent samples were used for the immediate Live/Dead cell viability assay and the histology. Figures 9a and 9b show the results from the Live/Dead assay immediately performed for the incubator-cultured construct and the e-incubator-cultured construct, respectively. Live cells are shown in green, and dead cells are shown in red color. Viability data for both constructs did not show significant differences. The histology performed using von Kossa staining revealed differentiation toward the human osteogenic lineage, as shown in Figs. 9c and 9d.

DISCUSSION

FIGURE 8. (a) Means and SDs of the local T2 values inside the ROI drawn on T2 map images to outline the construct in a 4-week study. (b) T2 relaxation maps (ms) generated weekly show a decrease from average of 143.4 6 6.1–64.1 6 6.4 ms during the 4-week study. The construct is inside the culture well. (c) ADC data were processed daily and associated average values were illustrated. (d) ADC maps generated for the e-incubator-cultured construct in four time points. The average ADC values showed a trend to decrease from 1.8 6 0.1 to 1.5 6 0.2 mm to 2/s 3 1023 during the study.

We have developed a high-field MRI compatible incubation platform, which enables repeated and noninvasive examination of the sample in a controlled environment and with minimum operator handling. Termed e-incubator, this system supervises the culture environment including temperature, pH, and CO2 concentration along with providing media exchange automatically. The temperature, CO2 and pH were within acceptable incubation criteria’s.

Medical Imaging-Compatible Autonomous Incubation System

An osteogenic construct was cultured inside the e-incubator for 4 weeks and compared with its counterpart cultivated in a conventional CO2 incubator in order to examine the long-term function of the system. We designed different types of multiple-well sample holders to incubate multiple constructs with different size and geometries simultaneously. Additionally, using a multiple-well sample holder, the e-incubator can be applied to study, for example, tumors by incubating tumor cells in one chamber and the target tissue in the other chamber. Various aspects of the culture can be characterized using different MR parameters such as global density, T1 and T2 relaxation times, ADC, and MTR. The e-incubator was designed as a novel autonomous incubation system for long-term cultures enabling us to appropriately track the culture using MRI. The obtained MR images and corresponding data represent the capability of the system to work as an in vivo mimicking environment that is observable in real-time by the imaging system. The viability test and bone formation, proved by histology, verified that the e-incubator can be efficiently be used to produce human tissue-engineered bone. It also allows us to noninvasively measure the properties of the construct and finally harvest it without exposing it to contamination or any source of stress. In this study we only measured the T2 relaxation time and ADC characteristics of the bone construct. These quantified MR parameters showed a very good agreement with the data presented by Xu et al.22 Their findings described that T2 and ADC for engineered osteogenic constructs over 4 weeks tissue development dropped 64 and 40% compared with control at week four. Based on this comparison, the e-incubator doesn’t compromise the quality of the MR measurements. In this study, T2 and ADC for the e-incubator cultured construct decreased 55 and 17% respectively. The T2 relaxation times in Xu et al.’s study were shorter due to the higher magnetic field of 11.7 T. These two parameters can be utilized as markers for the osteogenic differentiation of the human mesenchymal stem cells. As the next step, we will transform the e-incubator to an MRI imaging compatible bioreactor by applying mechanical and electrical stimulations to the cultured construct which have been shown to deliver significant influences on the culture.18 Furthermore, nesting mechanical actuators around the culture chamber will permit us to run the magnetic resonance elastography (MRE) experiments in order to measure the mechanical properties of the culture during growth.4,11 These modifications might distort the homogeneity of the static magnetic field to such a degree that an additional correction will be necessary through a modified pulse sequence.2 Moreover, contrast agents such as magnetic

nanoparticles (iron oxide particles) can be used to improve the contrast in the MRI image.20 This system has versatile potential applications in studies that need an environment that closely mimics required in vivo conditions such as regenerative medicine, drug delivery, and pathophysiology of the diseases, in particular, cancer treatment.

ACKNOWLEDGMENTS We acknowledge funding support from the Nebraska Stem Cell Grant (Stem Cell 2013–07). Additionally, we acknowledge the help and support of Dr. Karin Wartella and Dr. Shadi Othman. The authors kindly thank Melody A. Montgomery at the University of Nebraska Medical Center (UNMC) Research Editorial Office for the professional editing of this manuscript.

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