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, 10 (1), 4824

Large Scale and Integrated Platform for Digital Mass Culture of Anchorage Dependent Cells

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Large Scale and Integrated Platform for Digital Mass Culture of Anchorage Dependent Cells

Kyoung Won Cho et al. Nat Commun.

Abstract

Industrial applications of anchorage-dependent cells require large-scale cell culture with multifunctional monitoring of culture conditions and control of cell behaviour. Here, we introduce a large-scale, integrated, and smart cell-culture platform (LISCCP) that facilitates digital mass culture of anchorage-dependent cells. LISCCP is devised through large-scale integration of ultrathin sensors and stimulator arrays in multiple layers. LISCCP provides real-time, 3D, and multimodal monitoring and localized control of the cultured cells, which thereby allows minimizing operation labour and maximizing cell culture performance. Wireless integration of multiple LISCCPs across multiple incubators further amplifies the culture scale and enables digital monitoring and local control of numerous culture layers, making the large-scale culture more efficient. Thus, LISCCP can transform conventional labour-intensive and high-cost cell cultures into efficient digital mass cell cultures. This platform could be useful for industrial applications of cell cultures such as in vitro toxicity testing of drugs and cosmetics and clinical scale production of cells for cell therapy.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Large-scale, integrated, and smart cell culture platform. a Photograph showing LISCCP. Scale bar: 2 cm. b Schematic illustration of the ultrathin sensors and stimulators transfer-printed to the 3D engineered substrate to be integrated with the wireless control system (left) and the exploded view of the ultrathin device layers (right). cf Optical microscopic image of the impedance sensor/electrical stimulator (c), pH sensor (d), K+ sensor (e), and temperature sensor and heater (f). Scale bars for c, f: 200 μm; for d, e: 500 μm. g Photograph showing the five-layer stacked 3D-printed engineered PLA substrates that were designed with protruding hemispheres (D = 1 mm, inset), perforations (1 mm × 1 mm), and stackable pillars and holes and integrated with the ultrathin electronics. Scale bar: 1 cm. Inset shows the SEM image of the protruded hemispheres. Scale bar: 500 μm. h Photograph of the 3D stack assembly of three LISCCPs. Scale bar: 5 cm. i Photograph of a CO2 incubator filled with multi-stacked LISCCPs (a total of 10) for digital mass cell culture. Scale bar: 5 cm. j Photograph of two CO2 incubators filled with multi-stacked LISCCPs wirelessly integrated with a single laptop. Scale bar: 10 cm
Fig. 2
Fig. 2
Characterization of cell culture substrate and ultrathin sensors and actuators. a Photograph of stacked 3D-printed engineered substrates (side view, scale bar: 1 cm) and magnified view of the assembly region (inset, scale bar: 2 mm). b Plot comparing cell attachment on a stack of engineered substrates and on a single flat substrate. (n = 5, Box: median; 25th to 75th percentiles, Whisker: min to max, *P < 0.001 with two tailed unpaired t test). c Plot showing cell viability in each layer of the five-layer stack of engineered substrates and flat substrates. The number of the layers descends from the top layer. (n = 4, mean, *P < 0.001 versus flat substrate, ANOVA with Bonferroni’s post-test). d Plot showing the viability of cells based on the depth of culture medium (n = 3, mean ± s.d., *P < 0.001 compared to control (depth = 0 mm), ANOVA). e Image showing GO spray-coating onto the 3D printed PLA substrate. Inset shows a spray containing GO. f AFM image of the GO-coated PLA substrate. g SEM images of the GO-PLA substrate before and after cell seeding. Scale bar: 20 μm. h Plot showing the number of cells cultured on polystyrene, glass, and GO-coated PLA substrates (n = 4, mean, *P < 0.001, ANOVA with Bonferroni’s post-test, ns, not significant). il Characterization of sensors and stimulators. i The graphs show the open-circuit potential (OCP) measurements from pH sensor at the pH range of 4–10 (left) and in subtle changes between pH 6.95 and 7.95 compared to a commercial pH sensor (numbers on each step) (right). j The graphs show OCP measurements from K+ sensor at concentrations between 1 and 32 mM (numbers on each step) (left) and in subtle changes between 0.125 and 2 mM (right). The insets in i, j show calibration curves for both ion sensors (n = 5). k Current–voltage plot of electrical stimulator (left) and log–log plot of the impedance measurements at different frequencies (right). Insets show reproducibility (n = 16 for left and n = 4 for right). l Graphs showing the resistance of temperature sensor changes with temperature (left) and thermal stimulator response to sudden temperature variations (inset). Graph showing the relationship between heat and applied voltage (n = 16) (right)
Fig. 3
Fig. 3
Design of a wireless system and control algorithm. a Photograph of the data acquisition hardware. be Control flow chart of the impedance sensing (b), electrical stimulation (c), thermal stimulation and temperature sensing (d), and pH and K+ sensing (e). f, g Plots showing the DAC output for impedance measurement (red) and filtered signals (blue) of the adjustable frequency range (10–10,000 Hz). h Comparison of the impedance measurement between wired (left) and wireless (right; screenshot image of the software) recordings. i Plot of electrical stimulations controlled by the user interface software to specify the site of stimulations. j Plots to compare wired and wireless measurements of pH and K+ concentrations. k Infrared camera images of the heat generated and controlled by the thermal stimulators (TS) wirelessly. The number of TS to turn on can be controlled
Fig. 4
Fig. 4
Real-time monitoring of various cells cultured on a single-layer CCP. a Photographic image of four single-layer CCPs in an incubator, controlled wirelessly by a single laptop. b Screenshot images of the user interactive software for each mode of monitoring and stimulations: impedance sensing (top left), pH/K+ sensing (bottom left), electrical stimulation (top right), and temperature sensing/thermal stimulation (bottom right). c Monitored data (impedance, temperature, pH, and K+) for cultures of four types of cells (hDFB human dermal fibroblasts, hMSC human mesenchymal stem cell, C2C12 mouse myoblast, HL-1 mouse cardiac muscle cell; mean). In the C2C12 culture, the growth medium was changed to the differentiation medium on day 5 (arrow). dg Impedance mappings of hDFB (d), hMSC (e), C2C12 (f), and HL-1 (g). The blue color indicates the lowest impedance, whereas red color indicates the impedance in maximum. h Growths of four types of cells as determined by the WST-8 assay. The absorbance is increased as the number of cells is increased. i Fluorescent microscopic images of F-actin-stained C2C12 cells during proliferation (top, scale bar: 10 μm) and myosin heavy chain staining of C2C12 cells showing myotube formation of the cells during differentiation (bottom, scale bar: 50 μm). j Expression of the muscle-specific gene for myogenin (red line) and MyoD (blue line) in C2C12 cells (n = 3, mean ± s.d., myogenin compared to day 0, *P < 0.05, **P < 0.01; MyoD compared to day 0 #P < 0.05, ##P < 0.01, ANOVA). k Fluorescent microscopic images of F-actinstained HL-1 during proliferation (top, scale bar: 40 μm) and connexin 43 (Cx43) expression of the cells during differentiation (bottom, scale bar: 30 μm)
Fig. 5
Fig. 5
3D array monitoring and stimulation control of C2C12 culture with LISCCP. a Impedance mapping of C2C12 cells cultured on the five-layered LISCCP without stimulations. Confluence appears at day 7 (red) before the differentiation proceeds. b Corresponding 3D pH mapping to a. c Photograph of LISCCP integrated with a peristaltic pump for culture medium circulation. Scale bar: 5 cm. d Profiles of pH with and without circulation of culture medium (n = 4, mean ± s.d., *P < 0.01, **P < 0.001, ANOVA). e Photograph of 25 culture layers with a culture medium circulation unit. Scale bar: 5 cm. f Cell viability at each layer in the 25-layer LISCCP (n = 5, ns, no significant; ANOVA). g Impedance monitoring for C2C12 cells cultured under ES, TS, or ES+TS. No stimulation was applied for the control (n = 4, mean ± s.d., ES+TS compared to control, *P < 0.01, **P < 0.001; ES compared to control, #P < 0.01, ##P < 0.001, ANOVA). h Expression of the muscle gene markers, myogenin (left) and MyoD (right), during the differentiation period (n = 3, mean ± s.d., ES+TS compared to control, *P < 0.01, **P < 0.001; ES compared to control, #P < 0.001, ANOVA with Bonferroni’s post-test). i Fluorescent analysis of C2C12 cells cultured without (control) and with stimulations (ES, TS, ES+TS). F-actin is stained red on day 5, myotube in green on day 10, and nucleus in blue. Scale bars for F-actin: 10 μm and for myotube: 50 μm. j Impedance mapping of a five-layered LISCCP when ES and TS are applied only on the third layer. k Impedance profiles of C2C12 culture on five layers with ES and TS (*P < 0.001, L3 compared to L1; #P < 0.001, L2 compared to L1). l Cell growth profiles showing increased cell growth on the stimulated layer (n = 4, *P < 0.05; ***P < 0.001 versus layer 1 and layer 5; #P < 0.001 versus layer 2 and layer 4, &P < 0.05, ANOVA). m Myogenic gene expression in C2C12 during differentiation (n = 3). n Profiles of pH on five layers (n = 4, mean ± s.d.)
Fig. 6
Fig. 6
Applications of multilayerd LISCCPs. a Photographs showing a multilayered LISCCP used for in vitro drug toxicity testing of drugs and cosmetics. LISCCP is divided into two single-layer CCPs and one triple-layered CCP to conduct three different tests. Scale bar: 5 cm. b Testing to evaluate the effects of 7CZ NP-mediated ROS scavenging on HL-1 using single-layer CCPs. The impedance mappings of HL-1 culture along with its corresponding live (green)/dead (red) cell images are shown for the treatment of H2O2+7CZ NPs (top) and H2O2 only (bottom). Scale bar: 500 μm. c Triple-layered impedance mapping of HL-1 treated with H2O2 at one corner and 7CZ NPs at the opposite corner (left) and their live/dead cell images (right). Scale bar: 500 μm. d Viability of hDFB cultured on each layer of LISCCP at t = 0 (n = 8, mean ± s.d., ns, not significant, ANOVA). e Impedance mappings and live/dead cell images of hDFB treated with phosphate-buffered saline (PBS), H2O2, or one of the surfactants (ALS ammonium lauryl sulfate, BAC benzalkonium chloride, Tween TWEEN 60). Scale bar: 100 μm. f Viability and number of hMSC cultured on LISCCP for three passages (cell number, n = 8, *P < 0.001 versus passage 1, #P < 0.01 versus passage 2, ANOVA with Bonferroni’s post-test; cell viability, n = 4, Box: median; 25th to 75th percentiles, Whisker: min to max, ns, not significant, ANOVA). Large expansion of cells shows the utilization of LISCCP in large-scale cell manufacturing for cell therapy. g Caspase-3 gene expression of hMSC cultured on LISCCP for three passages (n = 3, ns, not significant, ANOVA with Bonferroni’s post-test). h Staining images of hMSC differentiated into osteogenic (left, Alizarin red O staining) and adipogenic (right, Oil red O staining) lineage cells. Red indicates osteogenic or adipogenic lineage cells. Scale bar: 50 μm. i Impedance monitoring of hMSC cultures that were induced to undergo osteogenic and adipogenic differentiation by medium change on day 7 (arrow) when the cells were 90% confluent

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