With advancements in human induced pluripotent stem cell (hiPSC) technology, there is an increasing demand for quality control techniques to manage the long-term process of target cell production effectively. While monitoring systems designed for use within incubators are promising for assessing culture quality, existing systems still face challenges in terms of compactness, throughput, and available metrics. To address these limitations, we have developed a compact and high-throughput lens-free imaging device named INSPCTOR. The device is as small as a standard culture plate, which allows for the installation of multiple units within an incubator. INSPCTOR utilises a large thin-film transistor image sensor, enabling simultaneous observation of six independent culture environments, each approximately 1 cm2. With this device, we successfully monitored the confluency of hiPSC cultures and identified the onset timing of epithelial-to-mesenchymal transition during mesodermal induction. Additionally, we quantified the beating frequency and conduction of hiPSC-derived cardiomyocytes by using high-speed imaging modes. This enabled us to identify the onset of spontaneous beating during differentiation and assess chronotropic responses in drug evaluations. Moreover, by tracking beating frequency over 10 days of cardiomyocyte maturation, we identified week-scale and daily-scale fluctuations, the latter of which correlated with cellular metabolic activity. The metrics derived from this device would enhance the reproducibility and quality of target cell production.
Materials and Methods
Fabrication of thin-film transistor image sensor panel
INSPCTOR's image sensor consists of a low-temperature poly-silicon thin-film transistor (LTPS-TFT) circuit and an amorphous silicon (a-Si) pin photodiode formed on a glass substrate (Fig.S1). Both the LTPS-TFT and the a-Si pin photodiode were fabricated using standard photolithography processes for liquid crystal display production. The LTPS-TFT channel layer (poly-Si) was deposited using chemical vapour deposition (CVD) and subsequently crystallised through an excimer laser annealing process. The a-Si pin photodiode was stacked on top of the TFT using CVD. The upper electrode of the a-Si pin photodiode is a transparent indium tin oxide electrode, allowing the photodiode to absorb light from above. The top layer is covered with a transparent acrylic resin for surface protection. The surface of the acrylic resin has been plasma-treated to improve the adherence of the cultured cells. All the above manufacturing processes took place at Kyocera Corporation.
Construction of the base module of INSPCTOR
The base module of INSPCTOR consists of an LED light source, a control board, a power board, a Li-ion battery, and a connection interface for the sensor module (Fig.S2). The LED light source utilises a planar light source commonly used in LCD displays. The control board is equipped with an integrated circuit comprising a Field Programmable Gate Array (FPGA), a Microcontroller Unit (MCU), and memory, and it can be connected to the sensor module via pogo pins. The control board performs various functions, such as receiving control signals from a PC, driving the sensor, reading light signals from the sensor, converting the light signals from analogue to digital, and transmitting the light signals to the PC. Communication with the PC is conducted via Bluetooth. The power board is connected to the control board through an FFC connector, manages the lithium-ion battery's voltage control, and supplies power to the control board and the light source. The outer frame of the base module is fabricated by machining polycarbonate. All the above manufacturing processes took place at Kyocera Corporation.
HiPSC culture
The hiPSC line 585A145 was maintained according to the methods described in a previous report.46 Specifically, 6-well culture plates were coated with 3 µg/ml iMatrix-511 (982021, MATRIXOME) in D-PBS for 1 hour at 37°C, and then hiPSCs were cultured in StemFit medium (AK02N, Ajinomoto). The medium was changed daily, and cells were passaged using TrypLE Select (A1285901, Thermo Fisher Scientific) every 4 days. Cell counting was performed using Countess (Thermo Fisher Scientific). At each passage, 2×105 cells were seeded. For the first 24 hours post-passage, 10 µM Y-27632 (259-00613, FUJIFILM Wako) was added to the StemFit medium. When culturing on INSPCTOR, the sensor was coated with 5 µg/ml iMatrix-511 in D-PBS for 1 hour at 37°C, and the desired number of cells was passaged. After the passage, cells were incubated in StemFit containing 10 µM Y-27632 for 24 hours, followed by daily medium changes without Y-27632.
Mesoderm induction
We used a base medium consisting of RPMI-1640 with HEPES (189-02145, FUJIFILM Wako) supplemented with B27 Supplement without insulin (A1895601, Thermo Fisher Scientific) and penicillin-streptomycin (168-23191, FUJIFILM Wako), designated as RPMI/B27-ins. On the second day after seeding hiPSCs on INSPCTOR, the culture medium was switched to RPMI/B27-ins with 6 µM CHIR99021 (038-23101, FUJIFILM Wako) to initiate mesoderm differentiation. Control samples were switched to RPMI/B27-ins without CHIR. The medium was not changed during the subsequent 48-hour observation on INSPCTOR.
Cardiomyocyte differentiation induction
Cardiomyocyte differentiation was induced based on temporal modulation of Wnt signalling in monolayer culture.47, 48 RPMI/B27-ins containing 50 µg/ml ascorbate is denoted as RPMI/B27-ins/asc. On day 0, the third day after hiPSC seeding, the medium was switched to RPMI/B27-ins/asc supplemented with 6 µM CHIR99021 to initiate differentiation. On day 1, the medium was changed to RPMI/B27-ins/asc. On day 3, for ventricular-like cardiomyocyte induction, the medium was switched to RPMI/B27-ins/asc containing 5 µM IWP-2 (034-24301, FUJIFILM Wako), 5 µM XAV-939 (247-00951, FUJIFILM Wako), and 1 µM BMS-453 (19076, Cayman Chemical).48, 49 For atrial-like cardiomyocyte induction, the medium was switched to RPMI/B27-ins/asc containing 5 µM IWP-2, 5 µM XAV-939, and 1 µM Retinoic acid (186-01114, FUJIFILM Wako).48-50 From day 5 onwards, both ventricular-like and atrial-like cardiomyocytes were cultured under the same conditions. On day 5, the medium was changed to RPMI/B27-ins/asc. Subsequently, the medium was changed every other day to RPMI-1640 with HEPES containing B27 Supplement (17504044, Thermo Fisher Scientific) and penicillin-streptomycin, which is denoted as RPMI/B27. On day 11 or 12, cells were detached using TrypLE Select and reseeded onto dishes or sensors coated with Matrigel (354277, Corning). After seeding, cells were incubated in RPMI/B27 containing 10% KnockOut Serum Replacement (10828010, Thermo Fisher Scientific) for 24 hours, followed by medium changes every other day with new RPMI/B27. For glucose-free medium, no glucose RPMI-1640 (185-02865, FUJIFILM Wako) containing B27 Supplement, 5 mM sodium DL-Lactate (L4263, Merck), and penicillin-streptomycin was used.
Drug treatment on cardiomyocytes
On day 23 of ventricular-like cardiomyocyte induction, cells were passaged using TrypLE Select and seeded at 2×105 cells per well on the sensor. After seeding, cells were cultured in RPMI/B27 containing 10% KnockOut Serum Replacement for 24 hours, followed by medium changes every other day with RPMI/B27. Drug response assays were conducted on day 30 of the induction. The medium was changed just before the experiment. Drug concentrations were adjusted by replacing half of the medium with a medium containing adequate concentrations of isoproterenol (I0260, Tokyo Chemical Industry) and propranolol (168-28071, Fujifilm). Measurements were taken 15 minutes after the medium change. All conditions were sequentially measured using the same sample, starting from the lowest to the highest concentration as follows: control, 10 nM Iso., 30 nM Iso., 100 nM Iso., 300 nM Iso., 300 nM Iso. and 300 nM Pro., 300 nM Iso. and 1 µM Pro., and 300 nM Iso. and 3 µM Pro.
Image processing and quantification
For confluency quantification, the Rolling Ball algorithm was used to obtain and subtract a baseline from the image, and the same threshold was manually set across all conditions. Regions of interest (ROI) were set within the well, excluding the chamber edges. The proportion of pixels with signals exceeding the threshold was calculated. For quantification of EMT during mesoderm induction, the Sobel filter was used to compute the first derivative and visualise the spatial gradient. ROIs were set within the well, excluding the chamber edges. The average value of the first derivative image in each ROI was divided by the mean brightness of the original image and then normalised. To quantify cardiomyocyte beating dynamics, low pass filtering was applied to the temporal intensity fluctuations in each pixel to remove high-frequency noise and identify the baseline. The change ratio from the baseline was calculated pixel by pixel to normalise the intensity fluctuations. A Fast Fourier Transform analysis was performed on the pixel-level intensity fluctuations, and the dominant frequency in the movie was analysed by averaging the results from all pixels. Peak detection was performed on the pixel-level intensity fluctuation to visualise the contraction conduction based on the differences in peak timing. All the processes are carried out using Fiji51 and Python 3.7.
Taishi Kakizuka, Tohru Natsume, Takeharu Nagai (2024) Compact lens-free imager using a thin-film transistor for long-term quantitative monitoring of stem cell culture and cardiomyocyte production., Lab on a chip, Volume 24, Number 24, pp. 5290-5303
Published in 2024 Dec 3 (Electronic publication in Dec. 3, 2024, midnight )
(Abstract) With advancements in human induced pluripotent stem cell (hiPSC) technology, there is an increasing demand for quality control techniques to manage the long-term process of target cell production effectively. While monitoring systems designed for use within incubators are promising for assessing culture quality, existing systems still face challenges in terms of compactness, throughput, and available metrics. To address these limitations, we have developed a compact and high-throughput lens-free imaging device named INSPCTOR. The device is as small as a standard culture plate, which allows for the installation of multiple units within an incubator. INSPCTOR utilises a large thin-film transistor image sensor, enabling simultaneous observation of six independent culture environments, each approximately 1 cm(2). With this device, we successfully monitored the confluency of hiPSC cultures and identified the onset timing of epithelial-to-mesenchymal transition during mesodermal induction. Additionally, we quantified the beating frequency and conduction of hiPSC-derived cardiomyocytes by using high-speed imaging modes. This enabled us to identify the onset of spontaneous beating during differentiation and assess chronotropic responses in drug evaluations. Moreover, by tracking beating frequency over 10 days of cardiomyocyte maturation, we identified week-scale and daily-scale fluctuations, the latter of which correlated with cellular metabolic activity. The metrics derived from this device would enhance the reproducibility and quality of target cell production.(MeSH Terms)