Article pubs.acs.org/Langmuir
Heat-Transfer-Method-Based Cell Culture Quality Assay through Cell Detection by Surface Imprinted Polymers Kasper Eersels,*,† Bart van Grinsven,‡ Mehran Khorshid,† Veerle Somers,§ Christiane Püttmann,∥ Christoph Stein,∥ Stefan Barth,∥ Hanne Dilien̈ ,‡ Gerard M. J. Bos,⊥ Wilfred T. V. Germeraad,⊥ Thomas J. Cleij,‡ Ronald Thoelen,†,# Ward De Ceuninck,†,# and Patrick Wagner†,# †
Hasselt University, Institute for Materials Research IMO, Wetenschapspark 1, Diepenbeek, Belgium Maastricht University, Maastricht Science Programme, PO Box 616, 6200 MD Maastricht, The Netherlands § Hasselt University, Biomedical Research Institute, School of Life Sciences, Hasselt University/Transnational University Limburg, Agoralaan - Building B, Diepenbeek, Belgium ∥ RWTH Aachen University Clinic, Institute of Applied Medical Engineering, Department of Experimental Medicine and Immunotherapy, Pauwelsstraße 20, D-52074 Aachen, Germany ⊥ Maastricht University Medical Center+, Department of Internal Medicine, Division of Haematology, Postbus 5800, 6202 AZ Maastricht, The Netherlands # IMEC vzw, IMOMEC Division, Wetenschapspark 1, B3590 Diepenbeek, Belgium ‡
S Supporting Information *
ABSTRACT: Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heattransfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time. bacteria,6 viruses,7 and erythrocytes.8 Current cell-detection platforms are based on fluorescent labeling,9 gel electrophoresis screening,10 fluorescence-activated cell sorting (FACS),11 centrifugal size separation,12 or dielectrophoresis.13 Biomimetic cell-detection platforms are usually combined with microgravimetrical14−16 and electronic read-out platforms.17,18 These techniques either require expensive equipment or should be used in a stable lab environment. Recently, a new versatile read-out method for bio(mimetic) sensors has been developed,19 the so-called heat-transfer
1. INTRODUCTION The development of specific synthetic receptors such as molecularly imprinted polymers (MIPs) that are able to detect a wide variety of targets has led to the development of numerous biomimetic sensor applications.1−3 Synthetic receptors mimicking the binding capacity of a natural receptor may have certain benefits over their natural counterparts including straightforward and low-cost synthesis that allows for mass production, improved thermal, chemical, and physical stability, reusability, and an unlimited shelf life.4 Since MIPs are usually aimed at detecting low-molecular-weight compounds, the concept has been extended toward surface imprinted polymers (SIPs). This technique has been used for the detection of many biological targets including proteins,5 © 2015 American Chemical Society
Received: November 26, 2014 Revised: January 26, 2015 Published: January 28, 2015 2043
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
(ZR-75-1s) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal calf serum and 1% penicillin/streptomycin. All chemicals were obtained from SigmaAldrich N.V. (Diegem, Belgium). Cells were passaged at a confluence of about 80%. Prior to imprinting and HTM, cells were washed six times in 1× PBS (phosphate buffered saline solution, Lonza Braine SA, Braine-l’Alleud, Belgium). Cell counting to determine the cell concentration in buffer medium was done using a hemocytometer (VWR International, Leuven, Belgium). 2.2. Preparation of Cell Imprinted Polyurethane Layers. Polyurethane layers were synthesized by dissolving 122 mg of 4,4′diisocyanatodiphenylmethane, 222 mg of bisphenol A, and 25 mg of phloroglucinol in 500 μL of anhydrous tetrahydrofuran (THF). All reagents were used as received and had a purity of minimally 99.9% (Sigma-Aldrich N.V., Diegem, Belgium). The prepolymerization mixture was stirred at 65 °C for 200 min under an inert nitrogen atmosphere up to the gelling point. Next, the polymer was diluted in a 1:5 ratio in anhydrous THF and spin-coated for 60 s at 2000 rpm onto 1 cm2 polished aluminum substrates. This resulted in polyurethane layers with an average thickness of 1.2 ± 0.1 μm as measured with a profilometer (Dektak3ST, Sloan Instruments Corporation, Santa Barbara, CA). In parallel, homemade polydimethylsiloxane (PDMS) stamps were covered with cells to stamp the cells into the spin-coated polyurethane layer. The stamps were made using the Sylgard 184 silicone elastomer kit (Malvom N.V., Schelle, Belgium). Cell suspensions in PBS (400 μL) were applied onto the stamps, and after 50 s of sedimentation time, the excess fluid was removed by spinning at 3000 rpm for 60 s, resulting in a dense monolayer of cells on the stamp surface. The cell-covered stamp was gently pressed (pressure of 70 Pa) onto the polyurethane layer and cured for 18 h at 65 °C under a nitrogen atmosphere. After curing of the polyurethane layer, the stamp was removed and the template cells were washed off the surface by 0.1% SDS (sodium dodecyl sulfate) and PBS, leaving behind selective binding cavities on the polyurethane surface. In this study, SIPs were created for both the original and the descendant ZR75-1 cell line. 2.3. STR DNA Profiling. DNA from the cell lines under study was isolated via the peqGOLD Tissue DNA Kit (Peqlab, Erlangen, Germany) according to the manufacturers’ instructions. DNAamplification and STR analysis were performed as described in the literature40 using an ABI PRISM 3730 Genetic Analyzer (Life Technologies, Darmstadt, Germany) and GenMarker software (Softgenetics, State College, USA). Obtained sequencing results were compared to STR data from the Deutsche Sammlung von Mikroorganismen and Zullkulturen (DSMZ, Braunschweig, Germany). 2.4. Sensor Setup. The sensor setup and its performance in cellbinding assays have been described in earlier work.26,27 The proportional-integral-derivative (PID) settings (P = 1, I = 8, D = 0) used were optimized in a previous study.41 Each cell exposure event consists of an infusion of cell solution into the measuring chamber (volume ∼110 μL) followed by a flushing step with PBS. The measuring chamber is filled with PBS prior to the measurement, and the thermal resistance is left to stabilize for 30 min. Cells are then introduced into the flow cell of the system at a flow rate of 2.5 mL min−1 (72 s, 3 mL cell suspension). The cells get in contact with the SIP, and the signal is left to stabilize again for 30 min. Subsequently, the system is flushed with PBS at a flow rate of 0.25 mL min−1 (12 min, 3 mL buffer) to remove any unbound cells from the SIP-covered aluminum chip. Finally, the signal is allowed to stabilize for another 30 min. 2.5. Microscopic Imaging and Cell Labeling. Microscopic imaging of the cell-imprinted polyurethane surfaces was performed with an inverted optical microscope Axiovert 10 (Carl Zeiss, Jena, Germany). All SIPs were imaged at magnifications of 5× and 50×. ImageJ 1.44P (National Institute of Health, Bethesda, MA) was used to determine the number of cell imprints per area unit on microscopic images of the SIPs. The average surface coverage of cell imprints on the polyurethane layer was calculated on the basis of cell imprint
method (HTM). This technique has been successfully used for the detection of single-nucleotide polymorphisms in DNA,20−22 low-molecular-weight compounds through MIPs,23,24 and the study of phase transitions in lipid vesicle membranes.25 In addition, the combination of HTM with SIPs has led to the development of a sensitive, straightforward, cheap, and fast biomimetic sensor for the detection of cancer cells in buffer fluid.26 Although the results presented in this work were very promising, the detection limit of the methodology needs to be improved when aiming at point-of-care diagnostics.27 However, in this Article, we will demonstrate that the technology can already be used in its current form as a tool for monitoring the quality of cell cultures. Since the establishment of the first human cell lines early in the 1950s, namely, HeLa cells, derived from an invasive cervical carcinoma,28 the use of cell cultures as models for the study of a wide variety of diseases has progressively increased.29 The quality of cell cultures in terms of genetic stability, crosscontamination, and cell viability is very important for the reliability of these studies. The cross-contamination among cell lines in culture is a persistent problem that has been observed in 16−35% of cases under study.30 Cross-contamination has been reported for cell lines from hematopoietic,31 prostate cancer,32 and esophagus carcinoma.33 In addition to crosscontamination, spontaneous mutations occurring in cell cultures can have a negative effect on the overall quality of a cell culture.34 This problem is most pronounced in cancer cell lines, since these cells are characterized by a particularly high mutation rate.35 The possibility of a diminished cell culture quality has shown to increase when cells are kept in culture for prolonged periods of time.36 Negative effects of overpassaging cells have been demonstrated for many cell lines.37,38 Since most of these adverse effects do not result in a phenotypically apparent change in the cells, most cell line providers recommend to limit the number of subculture passages.39 However, the development of short tandem repeat (STR) DNA profiling has made it possible to uniquely identify cell lines and compare them to the original cells isolated from the tissue of a single individual.29 This allows researchers to assess the quality of the cell lines they have in culture by a relatively simple molecular technique. Although STR DNA profiling is very sensitive and has contributed greatly to the development of more accurate assays for the detection of cross-contamination and genetic instability in cell lines, there are several drawbacks associated with the technique. It involves DNA isolation and fluorescent labeling, requires expensive equipment that has to be used inside a lab environment, and is typically slow (in the order of days), and the data interpretation requires some training. In this Article, we demonstrate a cell culture quality assay that offers a fast, label-free, user-friendly, and low-cost alternative for STR DNA profiling. Therefore, we assess if the proposed platform is able to discriminate between the breast cancer cell line ZR-75-1 (adherent growth, ZR-75-1a) and a descendant cell line growing in suspension after having been overpassaged. In a next step, the original ZR-75-1 cell line was cultured and passaged over a prolonged period of time and analyzed with the proposed assay at regular time intervals. The results were compared with a STR DNA profiling analysis.
2. EXPERIMENTAL METHODS 2.1. Culturing of Cells. The breast cancer cell line ZR-75-1 (ATCC CRL-150) and the descendant cell line growing in suspension 2044
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
Table 1. Overview of STR Profiling Results Obtained with the Original ZR-75-1 Cell Line (ZR-75-1a in the Table) and a Descendant Cell Line Originating from the Same Cell Line (ZR-75-1s)a alleles under study cell line
D5S818
D13S317
D7S820
D16S539
VWA
TH01
AM
TPOX
CSF1PO
result
DSMZ (ref) ZR-75-1a ZR-75-1s
13, 13 13, 13 11, 11
9, 9 9, 9 10, 11
10, 11 10, 11 11, 12
11, 11 11, 11 11, 13
16, 18 16, 18 14, 19
7, 9.3 7, 9.3 7, 9.3
X, X X, X X, Y
8, 8 8, 8 8, 11
10, 11 10, 11 10, 12
95−100% 55−60%
a The results are compared to STR results obtained by DSMZ (first row) and indicate that the ZR-75-1s cell line is indeed corrupted by effects originating from over-passaging.
Figure 1. Optical microscopy images at 5× (a and c; scale bar marks 200 μm) and 50× (b and d; scale bar marks 20 μm) magnification of a SIP imprinted for ZR-75-1a and ZR-75-1s cells, respectively. counts of three different samples for each type of SIP and five locations on each sample.
3.2. HTM Analysis of ZR-75-1a and ZR-75-1s Cell Lines. To verify whether or not the HTM setup was able to discriminate between ZR-75-1a and ZR-75-1s cells, polyurethane-covered aluminum chips were imprinted for both cell types, as described in section 2.2. Microscopic analysis of imprints for both cell lines revealed that very similar spherical imprints were formed with diameters of ±20 μm (Figure 1). The imprint surface coverage for both types of SIPs did not show any significant difference (22 400 ± 900 and 23 100 ± 1500 imprints cm−2, respectively), and an overall average imprint surface coverage of 22 750 ± 1200 imprints cm−2 was achieved with the cell imprinting procedure (±10% cell surface coverage). The backside of the chips was directly coupled to the copper heat provider to ensure optimal thermal contact. SIP layers were consecutively exposed to analogue and target cell solutions, respectively. Unbound cells were washed off by flushing the measuring chamber with PBS after each cell addition cycle. The volumes and flow rates used are described in section 2.4. The thermal resistance of the solid−liquid interface was monitored as a function of time by the HTM
3. RESULTS 3.1. STR DNA Profiling Analysis of ZR-75-1a and ZR75-1s Cell Lines. DNA samples were isolated from both the original ZR-75-1 cell line (adherent growth) and the descendant cell line growing in solution. DNA profiles were obtained, and the resulting STR data were compared to data from the DSMZ, as described in section 2.3. The results of this analysis are summarized in Table 1 and indicate that the original cell line obtained from ATCC, that displays adherent growth, is indeed the breast cancer cell line, as the STR analysis is in accordance with the reference data from DSMZ. The STR analysis obtained with the descendant ZR-75-1s cell line only shows a 55−60% match with the reference data. This implies that the observed phenotypical change in the behavior of the cells, evolving from an adherent to a solution growth pattern, is indeed caused by a diminished quality of the cell line due to cross-contamination or spontaneous mutations, induced by overpassaging the cell line. 2045
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
Figure 2. Time-dependent Rth response of a SIP imprinted with ZR-75-1a cells upon consecutive exposure to a cell solution in PBS containing ZR75-1s and ZR-75-1a cells, respectively; the flow cell was flushed with buffer in between both additions to regenerate the sensor surface (a). The change in thermal resistance in time of a SIP imprinted for ZR-75-1s cells in a similar experiment is also shown in part c. The corresponding resulting changes in Rth (ΔRth) upon cell binding and flushing of the flow cell are summarized in box charts, shown in parts b and d. Error bars indicate the standard deviation of the noise on the signal.
W. Flushing the flow cell with buffer causes the signal to return back to baseline for the analogue cells, while the signal for the target cells remains at an elevated value. 3.3. HTM-Based Cell Culture Quality Assay. To assess if the proposed methodology was able to monitor the quality of a cell culture in time, the ZR-75-1a cell line was cultured for a prolonged period of time and analyzed using HTM at given time intervals. Therefore, SIPs were created for ZR-75-1a cells at the start of a new cell culture. The quality of the cell culture was monitored in time by performing a cell binding experiment at passage numbers of 5, 10, 13, 15, 18, 20, 23, and 25. At each of these passage numbers, a subset of the cells in culture was washed and diluted in PBS to a concentration of 1 × 105 cells mL−1. Each experiment was performed as described in section 2.4. The results of these experiments are summarized in Figure 3; the individual measurements and raw data can be found in Figure S1 (Supporting Information). To faithfully compare the
setup (one measuring point per second), and the results of this analysis are shown in Figure 2. The time-dependent thermal resistance (Rth) data for a SIP imprinted with ZR-75-1a cells (Figure 2a and b) demonstrate that the signal increases upon addition of a solution of analogue cells in PBS (ZR-75-1s cells, concentration: 1 × 106 cells/mL) by 1.12 ± 0.2 °C/W. Upon flushing the flow cell with PBS, the signal drops back to a value of 0.11 ± 0.2 °C/W above the baseline. After infusing a target cell solution into the measuring chamber (ZR-75-1a cells, concentration: 1 × 106 cells/mL), the signal increases again by 1.13 ± 0.2 °C/W. However, flushing with buffer solution does not cause a measurable decrease in Rth, as the signal remains at 1.12 ± 0.2 °C/W above the baseline. The results obtained with a SIP imprinted with ZR-751s cells show the same behavior. Exposing the sample to a solution of analogue cells causes an increase in thermal resistance by 0.84 ± 0.2 °C/W, while exposing the SIP to target cells induces an increase in thermal resistance of 1.02 ± 0.1 °C/ 2046
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
Figure 3. Normalized, time-dependent Rth response of a SIP imprinted with ZR-75-1a cells at passage 1. Upon stabilization of the signal in PBS buffer, target cells are introduced at a flow rate of 2.5 mL min−1. Weakly bound cells are washed of the surface by rinsing the flow cell with PBS at 0.25 mL min−1. SIPs are exposed to ZR-75-1 cells at passage 5, 10, 13, 15, 18, 20, 23, and 25. The color code is stated in the figure legend. The spikes in the signal are induced by flushing in cells and buffer solutions at room temperature.
Figure 4. Schematic representation of the time-dependent Rth data for a typical cell exposure event. Upon addition of cell solutions in buffer into the flow cell of the setup, an initial increase in thermal resistance is observed (ΔRini th ). Flushing the flow cell with buffer solution results in the removal of any unbound or weakly bound cells, resulting in a ini net decrease of the signal to a lower value (ΔRnet th ). Both ΔRth and ΔRth are calculated using the mean value of the signal in a stable plateau using 300 data points.
data, each data set was normalized with respect to its baseline Rth signal. The results in Figure 3 indicate that the thermal resistance of the solid−liquid interface under study increases in each experiment due to infusion of the cell solution by about 20%. For cells at passage number 5, 10, 13, and 15, the signal remains at an elevated level even after rinsing the flow cell with PBS. However, starting from passage number 18, a decrease in the Rth signal is observed after the washing step, indicating that a subset of the cells is no longer bound tightly to the SIP layer. To determine the extent of this decrease and to visualize the effect that long-term passaging of ZR-75-1 cells has on the HTM analysis, we calculate the Rth response in terms of ini net ini percentage as %Rth = (ΔRnet th /ΔRth ) × 100%. ΔRth and ΔRth are defined as the difference in average Rth upon an entire celladdition experiment and cell exposure, respectively, as described schematically in Figure 4. The %Rth was calculated for all the experiments shown in Figure 3, and the resulting values are plotted as a function of the passage number under study in Figure 5. The resulting graph reveals that the signal remains at a maximal value, stable within noise levels of the system, during the first 15 cell culture passages. However, at passage 18, the signal decreases to 67.0 ± 9.2% of the initial value and a further decrease is observed at passage 20 (43.2 ± 10.1%) and passage 23 (21.7 ± 11.5%) after which no further decrease is observed at passage 25. These results indicate that the cells become more susceptible to cell removal by flushing. The good accordance with the sigmoidal fit (red curve, R2 > 0.99, fit function: y = [A1 + (A2 − A1)]/[1 + 10(LOGx0−x) *p]) indicates that after a certain passage number th the bonds formed between cells and imprint rapidly become weaker. 3.4. Validation of Cell Culture Quality Assay by STR DNA Profiling and HTM. DNA was extracted from the ZR75-1a cells at passage 25, used in the previous experiment, and a DNA profile was obtained using the procedure described in section 2.3. The results obtained were compared to reference
Figure 5. Time-dependent %Rth data of a HTM-based cell culture quality assay. The %Rth values are calculated using the data shown in Figure 2 and plotted as a function of the cell culture passage number under study. The error bars represent the relative standard error on the data.
data of the DSMZ and to the data obtained with the original ZR-75-1 cell line at the beginning of the experiment. The results are shown in Table 2. Overpassaging has led to changes in the DNA expression pattern, as evidenced by the results from the STR analysis (55− 60% match). In addition, the data reveal a perfect match with the profile for ZR-75-1s cells, indicating that overpassaging has indeed resulted in cross-contamination of the cells in culture. This assumption was further assessed by validating the results by HTM. To this extent, an aluminum chip was covered with polyurethane and imprinted for ZR-75-1s cells and the sample was exposed to a cell solution containing the cells at passage 23 in the same way as in the experiments described in section 2.6. 2047
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
Table 2. Overview of STR Profiling Results Obtained with the Original ZR-75-1a Cell Line (ZR-75-1a (passage 1) in the Table) and an over-Passaged Cell Line (ZR-75-1a (passage 25))a alleles under study cell line
D5S818
D13S317
D7S820
D16S539
VWA
TH01
AM
TPOX
CSF1PO
result
DSMZ (ref) ZR-75-1a (pass 1) ZR-75-1a (pass 25)
13, 13 13, 13 11, 11
9, 9 9, 9 10, 11
10, 11 10, 11 11, 12
11, 11 11, 11 11, 13
16, 18 16, 18 14, 19
7, 9.3 7, 9.3 7, 9.3
X, X X, X X, Y
8, 8 8, 8 8, 11
10, 11 10, 11 10, 12
95−100% 55−60%
a The results are compared to STR results obtained by DSMZ (first row). These results indicate that the over-passaged cell line has been contaminated with ZR-75-1s cells.
contamination or spontaneous mutation will not result in any phenotypical change. The results obtained in this Article show that it is possible to discriminate between ZR-75-1a and ZR-75-1s cells. The corruption of the cell line by cross-contamination or spontaneous mutation, resulting from overpassaging the cells, has led to changes in cell surface chemistry (the presence/ absence of certain types of proteins, the amount of the proteins expressed, or the presence of different glycosylation patterns on these proteins).26,27 This explanation is supported by the timedependent analysis of the thermal resistance of the SIP layer. Exposing a SIP to a cell solution in PBS will lead to an increase of the thermal resistance. The response is similar for target and analogue cells due to their highly comparable morphology. However, mechanical shear forces provided by rinsing the flow cell with buffer will easily remove the more weakly bound analogue cells from the receptor layer, as evidenced by the drop in Rth to baseline after a washing step. The target cells are more strongly bound to the SIP layer, as the morphological match is assisted by a functional complementarity between the cells and the microcavities on the SIP surface. As a result, the cells are retained on the SIP surface and the signal does not return back to baseline upon flushing the setup with buffer medium. However, it is possible to regenerate the sensor surface by flushing the system with SDS and PBS consecutively. Previous work has demonstrated that this procedure can be repeated for at least six cycles with fidelity.26 The proposed biosensor platform has also demonstrated its applicability in an assay for monitoring the quality of a ZR-751a cell culture in time. The results of this experiment show that, after a certain number of cell culture passages, the thermal resistance starts decreasing upon flushing the flow cell with buffer solution. This implies that long-term passaging induces a change in the membrane functionalities of a subset of the cells in culture, resulting in a weaker bond between these cells and the imprints in the SIP layer, making them susceptible to cell removal by mechanical friction. STR profiling revealed that this change was induced by cross-contamination with ZR-75-1s cells, as evidenced by the perfect match in the DNA profile. The good accordance with the sigmoidal fit shown in Figure 5 indicates that these ZR-75-1s cells quickly outnumber the ZR75-1a cells due to their increased growth rate, resulting in an exponential decrease in %Rth. This illustrates the risk long-term passaging and resulting cross-contamination imposes on the quality of a cell line. In this case, both cell lines were cultured at the same time for a prolonged period of time, which, even when working under sterile conditions, clearly imposes a threat on the quality of the cell culture. The additional HTM analysis shown in Figure 6 further confirms the theory, as exposing the ZR-75-1s imprint to cells in passage 23 leads to an irreversible increase in thermal resistance, indicating that the observed
The thermal resistance of the solid−liquid interface was monitored in time, and the results are shown in Figure 6.
Figure 6. Time-dependent Rth response of a SIP imprinted with ZR75-1s cells upon exposure to ZR-75-1a at passage number 23. Infusion of the cell suspension into the flow cell leads to an increase in thermal resistance that is not reversible by flushing the cell with buffer solution, indicating that the ZR-75-1a cell line has been cross-contaminated with ZR-75-1s cells.
The results in Figure 6 demonstrate an increase in thermal resistance by 0.74 ± 0.11 °C/W upon exposure of the SIP to the cell suspension. The signal does not return back to baseline upon flushing with PBS but stabilizes at a value of 0.67 ± 0.10 °C/W. The resulting %Rth of 91.9 ± 15% indicates that there is a nearly perfect match between the cells and the SIP. This confirms the theory that the cell line was cross-contaminated with ZR-75-1s cells.
4. DISCUSSION The optical microscopy analysis of imprints made by ZR-75-1a and ZR-75-1s cells reveals that both cell lines create microcavities that are similar in size and shape. This can be explained by the fact that, although the cell lines differ in their DNA expression profile, as evidenced by the STR analysis, their morphology is very similar. The only phenotypical difference between both cell lines is their growth pattern (adherent versus suspension growth), but this difference is not apparent when dislodging the cells for passaging/imprinting. This further emphasizes the need for an assay that is able to assess the quality of cell lines in a straightforward, low-cost, and fast manner that allows for continuous monitoring of cell lines in culture. In many cases, the differences induced by cross2048
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir
manuscript was jointly written by K.E. and B.v.G. All authors have given approval to the final version of the manuscript.
decrease in cell culture quality is indeed the result of crosscontamination with ZR-75-1s cells.
Notes
The authors declare no competing financial interest.
5. CONCLUSION The results presented in this study further confirm that cell culturing is a delicate process, and it is best to avoid growing multiple cell lines simultaneously. However, this is not always possible when working in a cell culture lab with many different cell biologists that are studying different types of cell lines. Therefore, it is recommended to discard the cell lines after a fixed number of cell passages (10−15 passage numbers) to minimize the risk for cross-contamination and their negative effects on the quality of a cell culture. In addition, this Article demonstrates that the combination of highly selective and specific synthetic cell receptors such as SIPs and the thermal read-out platform HTM has led to the development of a novel tool for cell culture quality assessment. Although HTM does not provide any dynamic information on the specific effects of overpassaging on the DNA expression of the cells and is limited to changes in the surface chemistry of the cell line under study, it offers many advantages over stateof-the-art techniques such as STR DNA profiling. HTM is a very low-cost technique that requires a minimal instrumentation (the total cost of the device is ∼€1000). In addition, HTM is a fast technique: an entire cell binding measurement can be performed in 1.5 h, and the resulting data can be analyzed in a straightforward, automated manner. Furthermore, the low-cost and fast nature of the assay is even more apparent due to the fact that it does not require DNA isolation and fluorescent labeling. Finally, the device does not require a controlled lab environment, as all measurements were performed “on-thebench”. These characteristics make HTM an elegant tool for continuously monitoring the quality of cell cultures and thereby improving the reliability of scientific studies based on these cell lines.
■
■
ACKNOWLEDGMENTS This work was financed by the Life Science Initiative of the Province of Limburg, the Special Research Funds BOF of Hasselt University, the Research Foundation Flanders FWO (projects G.0B.6213.N and “Methusalem Nano”), and the Interreg IV-A project MicroBioMed (European Funds for Regional Development). Technical support by J. Baccus, L. De Winter, J. Soogen, and J. Mertens in building the mechanical and electronic system components is greatly appreciated. The authors are also grateful to C. Bocken for valuable advice on handling, culturing, and isolating the human cell lines.
■
(1) Haupt, K.; Mosbach, K. Molecularly Imprinted Polymers and Their Use in Biomimetic Sensors. Chem. Rev. 2000, 100, 2495−2504. (2) Apodaca, D. C.; Pernites, R. B.; Ponnapati, R.; Del Mundo, F. R.; Advincula, R. C. Electropolymerized Molecularly Imprinted Polymer Film: EIS Sensing of Bisphenol A. Macromolecules 2011, 44, 6669− 6682. (3) Reddy, S. M.; Phan, Q. T.; El-Sharif, H.; Govada, L.; Stevenson, D.; Chaven, N. E. Protein Crystallization and Biosensor Applications of Hydrogel-Based Molecularly Imprinted Polymers. Biomacromolecules 2012, 13, 3959−3965. (4) Vlatakis, G.; Andersson, L. I.; Müller, R.; Mosbach, K. Drug Assay Using Antibody Mimics Made by Molecular Imprinting. Nature 1993, 361, 645−647. (5) Shi, H.; Tsai, W. B.; Garrison, M. D.; Ferrar, S.; Ratner, B. D. Template-imprinted nanostructured surfaces for protein recognition. Nature 1999, 398, 593−597. (6) Alexander, C.; Vulfson, E. V. Spatially Functionalized Polymer Surfaces Produced via Cell-Mediated lithography. Adv. Mater. 1997, 9, 751−755. (7) Hayden, O.; Lieberzeit, P. A.; Blaas, D.; Dickert, F. L. Artificial Antibodies for Bioanalyte Detection-Sensing Viruses and Proteins. Adv. Funct. Mater. 2006, 16, 1269−1278. (8) Hayden, O.; Mann, K. J.; Krassnig, S.; Dickert, F. L. Biomimetic ABO Blood-Group Typing. Angew. Chem., Int. Ed. 2006, 45, 2626− 2629. (9) Riethdorf, S.; Fritsche, H.; Müller, V.; Rau, T.; Schindlbeck, C.; Rack, B.; Janni, W.; Coith, C.; Beck, K.; Jänicke, F.; Jackson, S.; Gornet, T.; Cristofanilli, M.; Pantel, K. Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System. Clin. Cancer Res. 2007, 13, 920−928. (10) Hempe, J. M.; Craver, R. D. Quantification of Hemoglobin Variants by Capillary Isoelectric Focusing. Clin. Chem. 1994, 40, 2288−2295. (11) Hou, H. W.; Warkiani, M. E.; Khoo, B. L.; Liu, Z. R.; Soo, R. A.; Twan, D. S.; Lim, W. T.; Han, J.; Bhagat, A. A. S.; Lim, C. T. Isolation and Retrieval of Circulating Tumor Cells Using Centrifugal Forces. Sci. Rep. 2013, 3, 1259. (12) Jones, P. V.; DeMichele, A. F.; Kemp, L. K.; Hayes, M. A. Differentiation of Escherichia coli Serotypes Using DC Gradient Insulator Dielectrophoresis. Anal. Bioanal. Chem. 2014, 406, 183−192. (13) Fu, A. Y.; Spence, C.; Scherer, A.; Arnold, F. H.; Quake, S. R. A Microfabricated Fluorescence-Activated Cell Sorter. Nat. Biotechnol. 1999, 11, 1109−1111. (14) Hayden, O.; Bindeus, R.; Dickert, F. L. Combining Atomic Force Microscope and Quartz Crystal Microbalance Studies for Cell Detection. Meas. Sci. Technol. 2003, 14, 1876−1881. (15) Jenik, M.; Schirhagl, R.; Schirk, C.; Hayden, O.; Lieberzeit, P.; Blaas, D.; Paul, G.; Dickert, F. L. Sensing Picornaviruses Using
ASSOCIATED CONTENT
S Supporting Information *
Figure showing time-dependent thermal resistance data. This material is available free of charge via the Internet at http:// pubs.acs.org.
■
REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Address: Hasselt University, Institute for Materials Research, Wetenschapspark 1, B-3590 Diepenbeek, Belgium. Phone: +3211-26.88.12. Fax: +32-11-26.88.99. E-mail: kasper.eersels@ uhasselt.be. Author Contributions
K.E. and B.v.G. contributed equally to this work. K.E. prepared all cell-imprinted polymer layers and was responsible for culturing cells in cooperation with M.K., C.P., C.S., G.M.J.B., W.T.V.G., and V.S. The heat-transfer device was designed by B.v.G, R.T., and W.D.C., and all heat-transfer measurements were performed by B.v.G. in cooperation with K.E and M.K. Optical analysis of the samples was done by K.E. Biological assistance and guidance on possible medical/biotechnological applications was provided by C.P., C.S., S.B., G.M.J.B., R.T., W.D.C., and W.T.V.G., while H.D. and T.J.C. provided input on SIP synthesis. B.v.G. and K.E. interpreted the heat-transfer data in close cooperation with W.D.C., R.T., and P.W. The 2049
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
Article
Langmuir Molecular Imprinting Techniques on a Quartz Crystal Microbalance. Anal. Chem. 2009, 81, 5320−5326. (16) Trekker, J.; Leten, C.; Struys, T.; Lazenka, V.; Argibay, B.; Micholt, L.; Lambrichts, I.; Van Roy, W.; Lagae, L.; Himmelreich, U. Sensitive In Vivo Cell Detection Using Size-Optimized Superparamagnetic Nanoparticles. Biomaterials 2014, 35, 1627−1635. (17) Qi, P.; Wan, Y.; Zhang, D. Impedimetric Biosensor Based on Cell-Mediated Bioimprinted Films for Bacterial Detection. Biosens. Bioelectron. 2013, 39, 282−288. (18) Law, J. K. Y.; Susloparova, A.; Vu, X. T.; Zhou, X.; Hempel, F.; Qu, B.; Hoth, M.; Ingebrandt, S. Human T Cells Monitored by Impedance Spectrometry Using Field-Effect Transistor Arrays: A Novel Tool for Single-Cell Adhesion and Migration Studies. Biosens. Bioelectron. 2014, DOI: 10.1016/j.bios.2014.08.007. (19) van Grinsven, B.; Eersels, K.; Peeters, M.; Losada-Pérez, P.; Vandenryt, T.; Cleij, T. J.; Wagner, P. The Heat-Transfer Method: A Versatile Low-Cost, Label-Free, Fast and User-Friendly Readout Platform for Biosensor Applications. ACS Appl. Mater. Interfaces 2014, 6, 13309−13318. (20) van Grinsven, B.; Vanden Bon, N.; Strauven, H.; Grieten, L.; Murib, M. S.; Jimenez Monroy, K. L.; Janssens, S. D.; Haenen, K.; Schöning, M. J.; Vermeeren, V.; Ameloot, M.; Michiels, L.; Thoelen, R.; De Ceuninck, W.; Wagner, P. Heat-Transfer Resistance at SolidLiquid Interfaces: A Tool for the Detection of Single-Nucleotide Polymorphisms in DNA. ACS Nano 2012, 6, 2712−2721. (21) Bers, K.; van Grinsven, B.; Vandenryt, T.; Murib, M.; Janssen, W.; Geerets, B.; Ameloot, M.; Haenen, K.; Michiels, L.; De Ceuninck, W.; Wagner, P. Implementing Heat Transfer Resistivity as a Key Element in a Nanocrystalline Diamond Based Single Nucleotide Polymorphism Detection Array. Diamond Relat. Mater. 2013, 38, 45− 51. (22) Cornelis, P.; Vandenryt, T.; Wackers, G.; Kellens, E.; LosadaPérez, P.; Thoelen, R.; De Ceuninck, W.; Eersels, K.; Drijkoningen, S.; Haenen, K.; Peeters, M.; van Grinsven, B.; Wagner, P. Heat Transfer Resistance as a Tool to Quantify Hybdrization Efficiency of DNA on a Nanocrystalline Diamond Surface. Diamond Relat. Mater. 2014, 48, 32−36. (23) Wackers, G.; Vandenryt, T.; Cornelis, P.; Kellens, E.; Thoelen, R.; De Ceuninck, W.; Losada Pérez, P.; van Grinsven, B.; Peeters, M.; Wagner, P. Array Formatting of the Heat-Transfer Method (HTM) for the Detection of Small Organic Molecules by Molecularly Imprinted Polymers. Sensors 2014, 14, 11016−11030. (24) Peeters, M.; Kobben, S.; Jiménez-Monroy, K.; Modesto, L.; Kraus, M.; Vandenryt, T.; Gaulke, A.; van Grinsven, B.; Ingebrandt, S.; Junkers, T.; Wagner, P. Thermal Detection of Histamine with a Graphene Oxide Based Molecularly Imprinted Polymer Platform Prepared by Reversible Addition-Fragmentation Chain Transfer Polymerization. Sens. Actuators, B 2014, 203, 527−535. (25) Losada-Pérez, P.; Jiménez-Monroy, K. L.; van Grinsven, B.; Leys, J.; Janssens, S. D.; Peeters, M.; Glorieux, C.; Thoen, J.; Haenen, K.; De Ceuninck, W.; Wagner, P. Phase Transitions in Lipid Vesicles Detected by a Complementary Set of Methods: Heat-Transfer Measurements, Adiabatic Scanning Calorimetry and DissipationMode Quartz Crystal Microbalance. Phys. Status Solidi A 2014, 211, 1377−1388. (26) Eersels, K.; van Grinsven, B.; Ethirajan, A.; Timmermans, S.; Jiménez Monroy, K. L.; Bogier, J. F. J.; Punniyakoti, S.; Vandenryt, T.; Hendriks, J. J. A.; Cleij, T. J.; Daemen, M. J. A. P.; Somers, V.; De Ceuninck, W.; Wagner, P. Selective identification of macrophages and cancer cells based on thermal transport through surface-imprinted polymer layers. ACS Appl. Mater. Interfaces 2013, 5, 7258−7267. (27) Bers, K.; Eersels, K.; van Grinsven, B.; Daemen, M.; Bogie, J. F.; Hendriks, J. J.; Bouwmans, E. E.; Püttmann, C.; Stein, C.; Barth, S.; Bos, G. M.; Germeraad, W. T.; De Ceuninck, W.; Wagner, P. HeatTransfer Resistance Measurement Method (HTM)-Based Cell Detection at Trace Levels using a Progressive Enrichment Approach with Highly Selective Cell-Binding Surface Imprints. Langmuir 2014, 30, 3631−3639.
(28) Scherer, W. F.; Syverton, J. T.; Gey, G. O. Studies on the Propagation In Vitro of Poliomyelitis Viruses. IV. Viral Multiplication in a Stable Strain of Human Malignant Epithelial Cells (Strain HeLa) Derived from an Epidermoid Carcinoma of the Cervix. J. Exp. Med. 1951, 97, 695−710. (29) Reid, Y.; Storts, D.; Riss, T.; Minor, L. Authentic Authentication of Human Cell Lines by STR DNA Profiling Analysis. In Assay Guidance Manual; Sittampalam, G. S., Gal-Edd, N., Arkin, M., Eds.; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, MD, 2004. (30) Nelson-Rees, W. A. The Identification and Monitoring of Cell Line Specificity. Prog. Clin. Biol. Res. 1978, 26, 25−79. (31) Drexler, H. G.; Dirks, W. G.; MacLeod, R. A. F. False Human Hematopoietic Cell Lines: Cross-Contamination and Misinterpretation. Leukemia 1999, 13, 1601−1607. (32) Thompson, E. W.; Waltham, M.; Ramus, S. J.; Hutchins, A. M.; Armes, J. E.; Campbell, I. G.; Williams, E. D.; Thompson, P. R.; Rae, J. M.; Johnson, M. D.; Clarke, R. LCC15-MB cells are MDA-MB-435: A Review of Misidentified Breast and Prostate Cell Lines. Clin. Exp. Metastatis 2004, 21, 535−541. (33) Boonstra, J. J.; van der Velden, A. W.; Beerens, W. C. W.; van Marion, R.; Morita-Fujimura, Y.; Matsui, Y.; Nishihira, T.; Tselepis, C.; Hainaut, P.; Lowe, A. W.; Beverloo, B. H.; van Dekken, H.; Tilanus, H. W.; Dinjens, W. N. M. Mistaken Identity of Widely Used Esophageal Adenocarcinoma Cell Line TE-7. Cancer Res. 2007, 67, 7996−8001. (34) Kaden, D.; Gadi, I. K.; Bardwell, L.; Gelman, R.; Sager, R. Spontaneous Mutation Rates of Tumorigenic and Nontumorigenic Chinese Hamster Embryo Fibroblast Cell Lines. Cancer Res. 1989, 49, 3374−3379. (35) Watanabe, N.; Okochi, E.; Mochizuki, M.; Sugimura, T.; Ushijima, T. The Presence of Single Nucleotide Instability in Human Breast Cancer Cell Lines. Cancer Res. 2001, 61, 7739−7742. (36) Hughes, P.; Marshall, D.; Reid, Y.; Parkes, H.; Gelber, C. The Costs of Using Unauthenticated Over-Passaged Cell Lines: How Much More Data Do We Need? Biotechniques 2007, 43, 575−586. (37) Briske-Anderson, M. J.; Finley, J. W.; Newman, S. M. Influence of Culture Time and Passage Number on Morphological and Physiological Development of Caco-2 Cells. Proc. Soc. Exp. Biol. Med. 1997, 214, 248−257. (38) Yu, H.; Cook, T. J.; Sinko, P. J. Evidence for Diminished Function Expression of Intestinal Transporters in Caco-2-Cell Monolayers at High Passages. Pharm. Res. 1997, 14, 757−762. (39) Freshney, R. I. Culture of Animal Cells, A Manual of Basic Technique, 3rd ed.; Wiley-Liss: New York, 1993. (40) Krenke, B.E.; Tereba, A.; Anderson, S.J.; Buel, E.; Culhane, S.; Finis, C.J.; Tomsey, C.S.; Zachetti, J.M.; Masibay, A.; Rabbach, D.R.; Amiott, E.A.; Sprecher, C.J. Validation of a 16-Locus Fluorescent Multiplex System. J. Forensic Sci. 2002, 47, 773−785. (41) Geerets, B.; Peeters, M.; van Grinsven, B.; Bers, K.; de Ceuninck, W.; Wagner, P. Optimizing the thermal read-out technique for MIP-based biomimetic sensors: Towards nanomolar detection limits. Sensors 2013, 13, 9148−9159.
2050
DOI: 10.1021/la5046173 Langmuir 2015, 31, 2043−2050
