Rapid Detection of Mycoplasma-Infected Cells by an ssDNA Aptamer

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Rapid Detection of Mycoplasma-Infected Cells by an ssDNA Aptamer Probe Yanting Liu,†,‡ Wenqi Jiang,†,‡ Shuanghui Yang,†,‡ Jianzhong Hu,† Hongbin Lu,† Wei Han,‡ Jianguo Wen,‡ Zihua Zeng,‡ Jianjun Qi,‡ Ling Xu,‡ Haijun Zhou,‡ Hongguang Sun,§ and Youli Zu*,‡ †

Xiangya Hospital, Central South University, Changsha 410008, China Department of Pathology and Genomic Medicine, Houston Methodist Hospital, 6565 Fannin Street, Houston, Texas 77030, United States § The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China Downloaded via NOTTINGHAM TRENT UNIV on August 13, 2019 at 06:02:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Mycoplasmas are unique cell wall-free bacteria. Because they lack a cell wall and have resistance to β-lactam antibiotics, mycoplasma is the major pathogen that infects cultured cells in research laboratories. For rapid detection of mycoplasma-infected cells, we developed an ssDNA aptamer sequence composed of 40 nucleotides. Flow cytometry analysis showed that the synthetic aptamer probe selectively targeted mycoplasma-infected culture cells with high specificity identical to commercially available PCR-based assays. Additionally, fluorescent microscopy studies revealed that the aptamer probe rapidly stained mycoplasmainfected cells with higher sensitivity compared to Hoechst dye-mediated cellular DNA content stains. Moreover, confocal microscopy studies of trypsin-treated cells validated that the aptamer probes selectively targeted mycoplasma components on the surface of infected cells. Finally, preclinical studies of peripheral blood cells demonstrated that the aptamer probe was able to detect in vitro mycoplasma infection of primary lymphocytes. Taken together, these findings indicate that the aptamer probe will not only allow rapid detection of mycoplasma-infected culture cells for research purposes but also provide a simple method to monitor mycoplasma infection in primary cell products for clinical use. KEYWORDS: aptamer probe, cell culture, mycoplasma infection, primary lymphocytes, rapid detection

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lipoprotein,18,19 and the enzymatic assay.20 Although the PCRbased assay is highly sensitive and specific, it is time consuming and may not distinguish between living and dead cells or media contamination. In contrast, Hoechst dye staining can be rapidly performed and allows direct visualization of mycoplasma-infected cells. However, because the Hoechst dye stains all cellular DNA contents, it is not specific for mycoplasma and has low sensitivity because of the high background from cell nuclear staining. The ELISA is fast and relatively simple to perform but is primarily used for the detection of mycoplasma antibody in cultured cells and can only detect a limited range of species.9 Enzymatic conversion of ADP to ATP can be detected by luciferase-containing kits but requires expensive luminometric equipment and can generate false negatives due to impaired enzyme function.20 The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive and can be used to test different cell cultures on a regular basis. Therefore, a simple and specific

INTRODUCTION Healthy cell cultures are indispensable for biomedical research. Microbial contamination of cultured cells may adversely impact research results.1 Mycoplasmas are unique cell wallfree bacteria that are resistant to β-lactam antibiotics.2,3 Therefore, mycoplasma is a major pathogen that infects cultured cells in research laboratories because it is resistant to the first-line antibiotics.4 Unfortunately, mycoplasma contamination of cell cultures is often missed due to lack of visible signs, such as turbid culture media5−7 or morphological changes of cultured cells.8,9 A previous study of 451 human leukemia/lymphoma cell lines showed that 28% of them were infected with mycoplasma.10 Other studies reported that in research laboratories, mycoplasma contamination rates of cultured cells may be as high as 70%.11−13 Notably, mycoplasma contamination can jeopardize almost all aspects of cell biology and lead to erroneous experimental results or the loss of culture cell lines.14 Currently, commercially available methods to detect mycoplasma-infected culture cells include PCR-based assays of mycoplasma genetic products,15,16 nonspecific Hoechst dyemediated cellular DNA content staining,17 enzyme-linked immunosorbent assay (ELISA) to detect the mycoplasma © XXXX American Chemical Society

Received: March 25, 2019 Accepted: August 1, 2019

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DOI: 10.1021/acssensors.9b00582 ACS Sens. XXXX, XXX, XXX−XXX

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Figure 1. Development of aptamers for Jeko-1 lymphoma cells. (A) Cell-binding assays of the enriched ssDNA pool products post cell-based SELEX by flow cytometry. Maximal evolution of cell-binding capacity to Jeko-1 cells was observed after the 15th SELEX cycle, but no change in cell binding to control Jurkat cells was detected. (B) Sequencing results and grouping analysis of the 15th round SELEX pool products. (C) Aptamer A15-1 showed the highest binding capacity to Jeko-1 cells, and no aptamers reacted with control Jurkat cells. (D) Aptamer A15-1 binds Jeko-1 cells but not control Jurkat cells. (E) Aptamer A15-1 rapidly stained the Jeko-1 cells, reaching maximal binding in 10 min. (F) Fluorescence microscopy confirmed specific binding of aptamer A15-1 to Jeko-1 cells (red). (G) Aptamer A15-1 did not bind to control Jurkat cells. (H) Aptamer A15-1 selectively stained Jeko-1 cells (red) but did not react with prestained control Jurkat cells (green) in the same cell mixture. Scale bar = 20 μm.

peptides35 but also they can target complex structures, including viral particles,36 bacteria,37 and eukaryotic cells.38 Aptamers specific for viruses, vaccines, or bacterial Mycobacterium tuberculosis infection have been investigated for diagnostic and therapeutic purposes.39−43 Cell-based systematic evolution of ligands by exponential enrichment (SELEX) is a powerful approach to develop aptamers that specifically bind to whole living cells without prior knowledge of the molecular targets on the cell surface.44 The resulting aptamers can be used as probes to discriminate between different cells45 or as discovery tools

assay that can rapidly detect mycoplasma-infected cells is greatly needed for biomedical research and clinical use. Aptamers are a group of small molecular ligands composed of single-stranded DNA (ssDNA) or RNA oligonucleotides.21−23 Similar to protein antibodies, aptamers can form a well-defined spatial conformation and recognize their targets with high affinity and specificity.24−26 As “chemical antibodies”, aptamers can be chemically synthesized and easily conjugated with different functional molecules.27−31 As molecular probes, aptamers not only can bind to simple molecules such as ions,32 drugs,33 small molecules,34 and B


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Fluorescence Microscopy. CellMask plasma membrane stains were purchased from Thermo Fisher Scientific (Waltham, MA). To assess binding specificity (Figure 1F), Jeko-1 and control Jurkat cells were treated with Cy3-labeled aptamers A15-1 (100 nmol/L) at RT for 30 min and examined under a fluorescent microscope (Olympus America, Melville, NY). In addition, a cell mixture was prepared by mixing equal numbers of Jeko-1 and Jurkat cells (Figure 1H). For identification purposes, control Jurkat cells were prestained with a 1000× diluted CellMask green membrane stain at RT for 30 min. The cell mixtures were treated with Cy3-labeled aptamer A15-1 (100 nmol/L) in binding buffer at RT for 30 min. After washing twice, cell smears were prepared and examined under a fluorescent microscope. All experiments were repeated three times. Mycoplasma Detection. In PCR detection, the e-Myco mycoplasma PCR detection kit (v2.0) was purchased from iNtRON Biotechnology (Kirkland, WA). Cells (3 × 105) were harvested, washed twice by DPBS, and then extracted the DNA from those cells by the QIAamp DNA mini kit (Qiagen, Germantown, MD, USA), and the concentration of the DNA was measured by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and diluted to 30 ng/μL per sample for mycoplasma testing. Next, 18 μL of DNase/RNase-free water and 2 μL of DNA sample were added into the premixed PCR tube (e-Myco mycoplasma PCR detection kit) as the sample group. Alternatively, 2 μL of positive control and 18 μL of DNase/RNase-free water were added into the premixed PCR tube for positive control, and 20 μL of DNase/RNase-free water was added into the premixed PCR tube for the negative control. The solutions were mixed thoroughly, and PCR was run, with the cycles set as follows: step 1, 94 °C 1 min for initial denaturation; step 2, 94 °C 30 s for denaturation; step 3, 60 °C 20 s for annealing; step 4, 72 °C 1 min for extension, step 5, 72 °C 5 min for final extension. Steps 2−4 were repeated for 30 cycles, and then, 3% agarose gel (with 1:10,000 ethidium bromide) was used for electrophoresis to analyze the results. For Hoechst staining, the Hoechst 33258 dye was purchased from Sigma-Aldrich (St. Louis, MO), and cells were stained at 1 μg/mL for 30 min, then washed twice with DPBS, mounted on the slide alive, and immediately observed under a confocal microscope. All experiments were repeated three times. Laser Scanning Confocal Microscopy Imaging. Cells (3 × 105) in 200 μL of culture medium were prestained with CellMask Deep Red membrane stain (1000× dilution) for 1 h to track a cell membrane and then treated with the Hoechst dye (1 μg/mL) and Cy3-labeled aptamer A15-1 (100 nM) for 30 min at room temperature. The cells were then washed twice with DPBS, mounted on the slide alive, covered with the coverslip, and observed with the olympus FV1000 confocal microscope (Olympus Corporation, Japan). The Olympus FV10-ASW 4.2 software was used to process the confocal images. The Hoechst dye was excited at 405 nm, and the emission was recorded at 461 nm (blue fluorescence). Cy3 was excited with a 543 nm laser, and the emission was recorded at 567 nm (red fluorescence). CellMask Deep Red was excited with a 633 nm laser, and the emission was recorded at 664 nm (pseudo green color). For Z-stack imaging, images were acquired by scanning every 0.41 μm depth in an intended range, and 32 pictures were acquired. The images were stacked and reconstructed into a three-dimensional image by Olympus FV10-ASW 4.2 viewer software. Mycoplasma Treatment. The mycoplasma removal agent (MPA) was purchased from VWR (Radnor, PA). MPA was added to the mycoplasma-contaminated cell lines, the concentration was maintained at 1 μg/mL, and the cells were cultured under normal conditions for 10 days. The culture medium was changed every 3 days. Mycoplasma Contamination. Mycoplasma-positive cells were harvested and centrifuged at 800g for 10 min. The collected media were used as the contaminate reagent and kept at 4 °C for no more than 7 days. The clean cell lines were cultured in a 100 mm dish with 15 mL of culture media, and 1.5 mL of contaminate reagent was added to each clean cell line. The infected cells were then cultured for 3 days without changing the media to facilitate the infection, and the

to analyze the molecular basis of a specific disease or infection process.43 In this study, we conducted cell-based SELEX to develop aptamers specific for Jeko-1 lymphoma cells. Unexpectedly, due to cell mycoplasma contamination, we identified the ssDNA aptamer sequence A15-1, which selectively targeted mycoplasma-infected cells. The sensitivity and specificity of this synthetic aptamer probe to mycoplasma-infected cells were confirmed in both cultured cell lines and primary human lymphocytes. This study presents a new simple method for rapid detection of mycoplasma-infected cells for both research and clinical use.

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EXPERIMENTAL SECTION

Cell Culture. The following cell lines were used: Burkitt’s lymphoma CA46, Raji, Daudi, Mantle cell lymphoma Jeko-1, Maver-1, Mino, anaplastic large-cell lymphoma (ALCL) Karpas 299, SUDHL-1, Hodgkin lymphoma HDLM-2, KMH-2, T leukemia Jurkat, chronic myelogenous leukemia K562, erythroleukemia HEL, acute promyelocytic leukemia NB4, HL-60, and multiple myeloma MM1S. Dr. Mark Raffeld at the NIH Center for Cancer Research (Bethesda, MD) generously provided the ALCL Karpas 299 and SUDHL-1 cell lines. Dr. Barbara Savoldo at Baylor College of Medicine (Houston, TX) generously provided the Hodgkin lymphoma HDLM-2 and KMH-2 cell lines. The other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were cultured in a culture medium RPMI 1640 (Corning, Manassas, VA) containing 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) and 100 IU/mL penicillin− streptomycin (GE Healthcare, Pittsburgh, PA). RPMI 1640 was used as the washing buffer. Binding buffer was prepared by adding 1 mg/ mL bovine serum albumin (BSA; Thermo Fisher Scientific, Waltham, MA) and 0.1 mg/mL t-RNA (Sigma-Aldrich, St. Louis, MO) to RPMI 1640. The separated human primary T cells were cultured in a T-cell culture medium with 1:1000 interleukin-2 (Thermo Fisher Scientific, Waltham, MA). The T-cell culture medium RPMI 1640 (Corning, Manassas, VA) containing 10% human serum (Sigma-Aldrich), 1% GlutaMAX (Thermo Fisher Scientific), 1% HEPES (Sigma-Aldrich), and 0.1% β-mercaptoethanol (Sigma-Aldrich). The culture conditions were the same as those used for the other cell lines. Flow Cytometry Analysis. In the cell-binding assay, Cy3-labeled aptamers A15-1 or Cy3-labeled ssDNA library (negative control) was dissolved in binding buffer and incubated with 5 × 105 cells for 15 min at room temperature, and the concentration of aptamer and library is 100 nM. Cells were washed twice with DPBS. The cell binding was quantified with a FACScan cytometer (LSRII, BD Biosciences, San Jose, CA) by counting 10,000 events. The dissociation constants (KD) were calculated from the cell-binding results detected by flow cytometry. The fluorescence intensity at different incubation times was calculated by the geometric mean of the PE channel, and data were analyzed by FlowJo v10. For preclinical studies, human peripheral blood cells were isolated from healthy donors and washed twice with DPBS. Blood cells (1 × 106) were then incubated with 10 μg/mL FITC mouse anti-human CD3 antibody, 6 μg/mL Cy5.5 mouse anti-human CD45 antibody, 12 μg/mL APC mouse anti-human CD56 antibody, 5 μg/mL APC-Cy7 mouse anti-human CD19 antibody, and the Cy3-labeled A15-1 aptamer (100 nM) for 15 min in the dark at room temperature, and all antibodies were purchased from BD Biosciences (San Jose, CA). Lysing solution (1.5 mL, BD Bioscience, San Jose, CA) was added to lyse the red blood cells. After centrifuging at 200g for 5 min, the supernatant was discarded, and the sample was washed once with DPBS. The cells were resuspended in 500 μL of DPBS and then quantified with a FACScan cytometer (LSRII, BD Biosciences, San Jose, CA) by counting 10,000 events. The data were analyzed by FlowJo v10. All experiments were repeated three times. C


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Figure 2. Aptamer A15-1 selectively bound mycoplasma-infected cells. (A) Flow cytometry analysis showed that aptamer A15-1 selectively bound culture cells infected with mycoplasma. (B) Aptamer A15-1 did not react with mycoplasma-free culture cells. (C) Cell status of mycoplasma infection was confirmed by commercially available PCR-based assays. centrifuged at 400g for 40 min at 20 °C. The upper layer was carefully discarded without disturbing the mononuclear cell layer, then transferred it into a new tube, and washed with 25 mL of balanced salt solution. The cells were counted and centrifuged at 300g for 10 min at 20 °C. The cell pellet was then resuspended in 800 μL of buffer (PBS with 0.5% BSA, 2 mM EDTA, pH 7.2) and 200 μL of CD3 MicroBeads (human-lyophilized, Miltenyi Biotec, Auburn, CA) were added, mixed them well, and incubated for 15 min at 4 °C. It was washed by 5 mL buffer, centrifuged at 300g for 10 min, and

infection status was checked with the e-Myco mycoplasma PCR detection kit (v2.0) 10 days later. Cultivation was continued, and the media were changed every 2 days. Human T-Cell Separation. Buffy coat products of healthy human blood were ordered from the Gulf Coast Regional Blood Center (Houston, TX). The buffy coats were diluted with a balanced salt solution (PBS with 2 mM EDTA, pH 7.2) at 1:2.5, mixed thoroughly, carefully layered in 35 mL of this mixture into 15 mL Ficoll-Paque (Miltenyi Biotec, Auburn, CA) in a 50 mL conical tube, and then D


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fluorescence) in the same mixtures. Therefore, aptamer A15-1 was selected for further study, and the predicted structure is shown in Figure S1B. Surprisingly, detailed validation studies with multiple culture cell lines revealed that aptamer A15-1 was not cell typespecific, as it bound to HDLM-2, KMH-2, K562, and HEL cells, but did not react with Maver-1, Daudi, SHUDHL-1, or HL60 cells (Figure 2A,B). The cell-binding patterns of aptamer A15-1 were analyzed and appeared unclassifiable based on the tested cell types. To identify the potential binding target of aptamer A15-1 on cells, we performed quality control studies to rule out culture contamination, including mycoplasma infection (Figure 2C), which has been reported to occur in 15 to 70% of cultured cells in research laboratories.13 Interestingly, all the tested cells, regardless of cell type, that were bound by aptamer A15-1 were infected by mycoplasma. Conversely, all nonreactive cells, again regardless of cell type, were mycoplasma-free, according to commercially available PCR assays (Figure 2C and Table 1).

resuspended in 2 mL buffer. Magnetic separation was performed with the auto MACS pro separator (Miltenyi Biotec, Auburn, CA). Cell Pellet Scanning. Freshly cultured mycoplasma positive cells (HDLM-2, KMH-2, and Jeko-1) and the same mycoplasma removed clean cells (2 × 106 cells/test) were harvested, diluted in 100 μL of binding buffer, and stained with 50 nM Cy3-labeled aptamer A15-1 (RT, 15 min). Cells were then washed twice with 1 mL of washing buffer and centrifuged at 500g for 5 min. The cell pellets were scanned by the XENOGEN IVIS 200 imaging system (PerkinElmer) at the DsRed channel (excited at 535 nm, emission at 620 nm) to detect the fluorescent signal. All experiments were repeated three times. Mycoplasma Species Sequence Analysis. Freshly cultured mycoplasma-contaminated Jeko-1 cells were maintained without a medium change for 3 days. Cells (1 × 106) were sent to the IDEXX BioAnalytics Company (Columbia, MO) for 16S rRNA sequencing. The resultant sequence data was compared to sequences in GenBank using BLAST software to determine mycoplasma species.

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RESULTS AND DISCUSSION Identification of the Aptamer Sequence for Mycoplasma-Infected Cultured Cells by Cell-Based SELEX. To develop aptamer(s) specific for mantle cell lymphoma, cultured Jeko-1 cells were used for cell-based SELEX, and cultured Jurkat cells (a T-cell lymphoma cell line) were used for counterselection, as shown in Figure S1A. After the 4th, 8th, 12th, 15th, and 17th rounds of SELEX, the resulting product pools were amplified and labeled with Cy3 fluorochrome, and cell binding was examined by flow cytometry analysis. Figure 1A shows that the binding capacity to Jeko-1 cells gradually increased from the 4th SELEX round and reached a maximum after the 15th SELEX round. In contrast, the selected products did not react with Jurkat cells in the control experiments. Next, the product pool after the 15th SELEX round was sequenced. Of the 150,393 total reads, the top 10 sequences were selected; their core sequences are listed in Figure 1B. Sequence grouping analysis revealed that aptamers A15-1 and A15-3 differed by only a single base and accounted for more than 31% of the total reads. A second group of aptamers (A152, A15-7, A15-9, and A15-10) accounted for approximately 17% of the total reads. In contrast, aptamer sequences of A154, A15-5, A15-6, and A15-8 were largely different and could not be grouped, although they accounted for more than 2% individually. Aptamer sequences A15-1, A15-2, A15-4, and A15-5 were synthesized and labeled with the Cy3 fluorescent reporter to evaluate their cell-binding capacity. Cultured cells were treated with synthetic aptamer probes, and the resulting cell-binding abilities of the aptamers were quantitatively analyzed by flow cytometry. As shown in Figure 1C, among the synthetic probes, aptamer A15-1 had the greatest binding ability to Jeko1 cells, while none of them reacted with control Jurkat cells. The binding affinity of aptamer A15-1 for Jeko-1 cells was high (KD = 24.5 nM), with a little reaction to control Jurkat cells even at a concentration of 500 nmol (Figure 1D). Notably, aptamer A15-1 rapidly targeted Jeko-1 cells and reached maximal cell binding in 10 min (Figure 1E). Resultant specific binding of aptamer A15-1 to Jeko-1 cells, but not to control Jurkat cells, was directly observed under a fluorescent microscope (Figure 1F,G). For validation, mixtures of Jeko-1 cells and prestained Jurkat cells were generated and then treated with aptamer A15-1. Figure 1H shows that aptamer A15-1 selectively stained Jeko-1 cells (red fluorescence) but did not react with the prestained control Jurkat cells (green

Table 1. Relationship between Aptamer Binding and the Mycoplasma Infection cell type Burkitt’s lymphoma

mantle cell lymphoma

anaplastic large-cell lymphoma Hodgkin lymphoma T leukemia chronic myelogenous leukemia erythroleukemia acute promyelocytic leukemia multiple myeloma

cell name

aptamer binding

mycoplasma PCR assay

CA46 Raji Daudi Jeko-1 Maver-1 Mino K299 SUDHL-1 HDLM-2 KMH-2 Jurkat K562

− − − + − − + − + + − +

− − − + − − + − + + − +

HEL NB4 HL-60 MM1S

+ − − −

+ − − −

Aptamer A15-1 Probe Is Both Specific and Sensitive in Detecting Mycoplasma-Infected Cells. First, mycoplasma-infected cells (HDLM-2 and KMH-2) were treated with the mycoplasma removal agent (MPA), as illustrated in Figure 3A. Elimination of mycoplasma from culture cells was monitored by PCR assays at days 3, 7, and 10 posttreatment (Figure 3B). Cell binding of aptamer A15-1 was simultaneously measured by flow cytometry at the same time points. As shown in Figure 3C, A15-1 cell binding decreased at day 3 posttreatment and was completely lost at days 7 and 10. These results were identical to the cell mycoplasma infection status detected in Figure 3B. On other hand, the noninfected cells (Daudi, HL-60, CD46, and Maver-1) were cultured in fresh mycoplasma-contaminated media for 10 days (Figure 3D). Resulting cell mycoplasma infection was then confirmed by PCR assays (Figure 3E). Staining of cells by aptamer A15-1 pre- and post-mycoplasma infection demonstrated that A15-1 specifically bound mycoplasma-infected cells but did not react with uninfected cells (Figure 3F). These findings demonstrated the specificity of A15-1 for mycoplasma-infected cells. E


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Figure 3. Aptamer A15-1 was specific for mycoplasma-infected cells. (A) Schematic of cell treatment to eliminate mycoplasma infection. (B) Post treatment, mycoplasma-free status was confirmed by PCR. (C) Cells pre- and posttreatments were stained with aptamer A15-1, and cell status of mycoplasma infection was quantified by flow cytometry analysis. (D) Schematic diagram of cell infection by mycoplasma. (E) PCR assay confirmed mycoplasma infection of cultured cells. (F) Flow cytometry analysis using aptamer A15-1 detected mycoplasma infection.

Figure 4. Aptamer A15-1 sensitively detects mycoplasma components of infected cells. (A) Mycoplasma-infected cells (HDLM-2 and Jeko-1) were prestained to track cell membrane (green signal) and simultaneously treated with aptamer A15-1 and Hoechst dye for DNA contents of both the mycoplasma and the nucleus. Confocal microscopy shows that aptamer A15-1 sensitively highlighted only mycoplasma components (red signal). (B) Under the same conditions, aptamer A15-1 did not react with cultured Maver-1 or CA46 cells that were mycoplasma-free. Scale bar = 10 μm.

For direct visualization and detection of mycoplasmainfected cells, the Hoechst dye has been widely used to stain cellular DNA contents.9 To compare the sensitivity of aptamer A15-1 with the standard Hoechst dye, mycoplasma-infected culture cells were simultaneously treated with both Cy3-tagged

aptamer A15-1 and the Hoechst dye. For tracking purposes, cell membranes were prestained in green (pseudo color). Posttreatment, cells were then examined under the confocal microscope. As shown in Figure 4A, aptamer A15-1 specifically highlighted mycoplasma components on the cell surface (red F


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Figure 5. Aptamer A15-1 targeted mycoplasma components on infected cells. (A) Schematic showing trypsin treatment of mycoplasma-infected cells. (B) Flow cytometry analysis of mycoplasma-infected cells (HDLM-2 and KMH-2) pre- and post-trypsin treatment. (C) The same sets of treated cells were simultaneously stained with aptamer A15-1 (red) and Hoechst dye (blue) and examined with confocal microscopy. Cell membranes were prestained (green). Complete elimination of mycoplasma components from the cell surface was validated by aptamer A15-1 stain (red). Scale bar = 10 μm.

Binding Targets of Aptamer A15-1 Are Mycoplasma Components on the Cell Surface. Mycoplasmas adhere to the surface of mammalian cells via a group of membranebound adhesins, such as P1, P30, P110, P116, P140, or HMW1-3.46,47 The lack of a mycoplasma cell wall enables direct contact of the mycoplasma membrane adhesins with the specific receptors on the host cell membrane.48,49 If we can remove the adhesins from the mycoplasma or the receptors from the host cell membrane, we can release the mycoplasma from the host cell membrane. Trypsin is a serine protease from

signal) without background cellular staining. In contrast, the Hoechst dye stained DNA contents of both the cell nucleus and mycoplasma components along with the cell membrane (blue signal). A strong background nuclear signal significantly reduced the sensitivity of the Hoechst dye stain. The same sets of cell stains were performed in control experiments with mycoplasma-free cells (Figure 4B). Aptamer A15-1 did not generate nonspecific signals, while the Hoechst dye caused a high background nuclear signal. G


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Figure 6. Monitoring mycoplasma infection of primary human cells by aptamer A15-1. (A) Primary T cells were isolated from the peripheral blood of healthy donors and cultured in the mycoplasma-contaminated medium for 7 days. Mycoplasma infection status of primary T cells was confirmed by PCR assays. (B) Flow cytometry analysis of primary T cells, pre- and post-mycoplasma contamination, using aptamer A15-1 detected the induced mycoplasma infection. (C) Mycoplasma infection of primary T cells was also detected and visualized using confocal microscopy with aptamer A15-1 stain. Scale bar = 5 μm.

the PA clan superfamily50 and can be used to cleave and identify membrane-bound proteins.51 To determine the binding targets of A150-1 on the cell surface, we treated the mycoplasma-infected cells (HDLM-2 and KMH-2) with trypsin, as illustrated in Figure 5A. Cells were then stained with aptamer A15-1, and changes in cell binding of A15-1 were determined by flow cytometry. Figure 5B shows complete loss of cell binding by A15-1 post-trypsin treatment, indicating that the mycoplasma components on the cell surface were the targets of aptamer A15-1. For direct visualization, the same set of cells, with or without trypsin treatment, was stained with the Hoechst dye and aptamer A15-1 simultaneously. For tracking purposes, the cell membrane was also stained. Merged confocal microscopy images confirmed complete elimination of cell surface mycoplasma components post-trypsin treatment (Figure 5C). Moreover, three-dimensional confocal imaging also confirmed that aptamer A15-1 specifically targeted mycoplasma components on the cell surface (Video S1). Utility of Aptamer A15-1 To Monitor Mycoplasma Infection of Primary Human Cells. In clinical settings, primary human cells are often used (e.g., in in vitro engineering and expansion of chimeric antigen receptor T cells [CAR T cells] for immunotherapy).52 To ensure the safety of primary cell products, it is important to monitor and rule out contaminating mycoplasma infection in cell cultures.11−13 To test the clinical utility of aptamer A15-1 in identifying mycoplasma infection, primary human T cells were isolated

from peripheral blood samples of healthy donors using CD3 MicroBeads.53 Primary T cells were then cultured in mycoplasma-contaminated media for 7 days, and the subsequent mycoplasma infection was confirmed by PCR (Figure 6A). The same set of cells was also stained with aptamer A15-1. Flow cytometry analysis revealed that aptamer A15-1 specifically stained mycoplasma-infected cells but did not react with uninfected primary T cells (Figure 6B). In addition, primary cells were simultaneously treated with the Hoechst dye and aptamer A15-1 post membrane staining. Merged confocal microscopy images validated that aptamer A15-1 was specific and sensitive to detect mycoplasma components on the infected cells but did not cause any nonspecific background signal on healthy primary T cells (Figure 6C). Finally, to evaluate the potential clinical value of aptamer A15-1, normal peripheral blood cells were stained with aptamer A15-1, as well as antibodies for cell identification. Different cell populations were separated by flow cytometry and gated based on FSC/SSC, CD45/SSC, CD3/CD56, and CD3/CD19 staining panels (Figure S2A). The gated individual cell populations were then analyzed for aptamer A15-1 staining. Figure S2B shows that aptamer A15-1 did not cause nonspecific binding to T cells, B cells, NK cells, monocytes, or eosinophils. Notably, a scant population of granulocytes showed low-level detection with aptamer A15-1, but the significance of this finding is unclear and is currently H


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Figure 7. Rapid detection of mycoplasma-infected cells by aptamer A15-1. (A) Paired mycoplasma negative and positive cells (HDLM-2, KMH-2, and Jeko-1) were treated with aptamer A15-1 probes, and cell pellets were then scanned using the IVIS 200 imaging system. (B) Bright light view of cell pellets. (C) Detection of mycoplasma-infected cell pellets under fluorescence view.

being investigated. Taken together, these studies demonstrate the potential clinical value of aptamer A15-1 to detect and/or monitor mycoplasma infection of primary cell products. Rapid Detection of Mycoplasma-Infected Cells. To show rapid detection of mycoplasma-infected cells can be achieved, we used mycoplasma-positive HDLM-2, KMH-2, Jeko-1 cells, and the same mycoplasma-negative cells and stained them with Cy3-labeled aptamer A15-1 (Figure 7A). After washing, cells were centrifuged to form cell pellets and then scanned by the IVIS 200 imaging system. The mycoplasma-positive cells showed a strong signal, but no signal was detected in the mycoplasma-negative cells (Figure 7B).

scleroderma patients and can induce scleroderma-like autoimmune disease after injection into mice.54 Thus, the development of aptamer A15-1 may offer a new diagnosis and/or treatment approach for scleroderma patients. This new aptamer probe will not only allow rapid detection of infected culture cells in research laboratories (Figure 7) but also provide a simple method to monitor mycoplasma infection in primary cells. The preclinical studies showed that this aptamer was able to detect primary human T cells that were infected by mycoplasma (Figure 6) and did not generate nonspecific staining in any population of tested blood cells (Figure S2). These findings indicate that aptamer A15-1 can be used to monitor mycoplasma contamination of clinical primary cell products, such as CAR T-cell generation for immunotherapy. Interestingly, since this aptamer specifically targets mycoplasma components on the cell surface, it is possible that an aptamer-drug conjugate could be useful for selective delivery into and/or targeted therapy of the infected cells. Moreover, the aptamer binding to mycoplasma components may form physical surface barriers and thus, prevent further infection. Therefore, identification of this mycoplasma-specific aptamer may open avenues for detection and prevention of mycoplasma infection and precision therapy via specifically targeting mycoplasma components.

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CONCLUSIONS An accidental mycoplasma contamination of cells produced completely different results that were anticipated and provided a great lesson to the aptamer research community: the optimal cell status and cultures free of microbial contamination are essential for success in cell-based SELEX. To identify mycoplasma species, infected culture cells were sequenced by the IDEXX BioAnalytics Company (Columbia, MO), and the results are shown in Table S1. Mycoplasma species analysis using 16s rRNA sequence data revealed that the contaminating species was Mycoplasma hyorhinis (Table S1). A study found that M. hyorhinis has been isolated from I


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00582. Three-dimensional view of A15-1 aptamer staining the mycoplasma infected cells (MP4) Experimental sections; cell-based SELEX and identified aptamer A15-1; utility of aptamer A15-1 to stain blood cells; aptamer A15-1 selectively bound mycoplasmainfected suspension cells and adhesion cells; mycoplasma species sequence analysis; three-dimensional view of A15-1 aptamer staining the mycoplasma infected cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yanting Liu: 0000-0001-6795-8545 Hongbin Lu: 0000-0001-7749-3593 Author Contributions

Y.L. performed the experiments, generated, and analyzed the data. Y.Z. designed experiments and revised the manuscript. W.J., S.Y., J.H., H.L., W.H., J.W., Z.Z., J.Q., L.X., and H.S. provided assistance in materials, experiments, and manuscript preparation. All authors approve the version of the manuscript to be published. Funding

This study is partially supported by NIH grant R01 CA224304. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Drs. Helen Chifotides, Sasha M. Pejerrey, and Kathryn E. Stockbauer for their scientific editing of this manuscript. Vasquez Matthew and ACTM Core Lab at Methodist Research Institute were acknowledged for the training and assistance of the confocal laser microscope.

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Rapid Detection of Mycoplasma-Infected Cells by an ssDNA Aptamer

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