High-Throughput Manipulation of Circulating Tumor Cells Using a

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High-throughput manipulation of circulating tumor cells using a multiple single-cell encapsulation system with a digital micromirror device Ryo Negishi, Kaori Takai, Tsuyoshi Tanaka, Tadashi Matsunaga, and Tomoko Yoshino Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00896 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Analytical Chemistry


High-throughput manipulation of circulating tumor cells using a multiple single-cell encapsulation system with a digital micromirror device Ryo Negishi, Kaori Takai, Tsuyoshi Tanaka, Tadashi Matsunaga, Tomoko Yoshino*.

Division of Biotechnology and Life science, Institute of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo, 184-8588, Japan.

*Corresponding author. Fax: +81-42-385-7713. Phone: +81-42-388-7021. E-mail: [email protected]

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic

information obtained from CTCs have the potential to provide new insights into the biology of

cancer metastasis. We previously developed a technique for single-cell manipulation based on

the encapsulation of a single cell in a photopolymerized hydrogel that can be used for

subsequent genetic analysis. However, this technique has limitations in terms of throughput

because light irradiation must be performed on each individual cell from the confocal

laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique

using a multiple single-cell encapsulation system with a digital micromirror device. This system

enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs

from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels

photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling

using two differently-shaped hydrogels was examined to distinguish between NCI-H1975 cells

and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In

addition to CTCs, our system can be widely applied for analyses of mammalian cells and

microorganisms.


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Introduction

Circulating tumor cells (CTCs) are defined as tumor cells that detach from the

primary tissue and enter the bloodstream.1-3 The concentration of CTCs in peripheral blood

samples can be used to predict the prognosis of patients with metastatic breast cancer, colorectal

cancer, and prostate cancer.4-6 Thus, CTCs are recognized as a useful and accessible biomarker

for cancer diagnosis. Recently, in addition to counting the number of CTCs, the molecular

characterization of CTCs—including genome and transcriptome analysis—has been

demonstrated, and the potential to provide detailed clinical information and new insights into

the biology of cancer metastasis has been shown.7-10 In addition, as it is widely known that

CTCs are genetically heterogeneous,11-13 the molecular characterization of CTCs should be

performed at the single-cell level. In fact, recent studies have identified inter- and intra-patient

heterogeneity in the mutational status of CTCs in metastatic breast and lung cancers.14-15 These

studies also reported that some CTCs have variants related to drug resistance and are potential

novel therapeutic targets. Thus, genomic analysis of single CTCs has the potential to be widely

applicable in personalized medicine and drug discovery.



For single-cell analysis of CTCs, techniques for recovering CTCs from blood

samples and the manipulation of single CTCs for subsequent analysis are required because

CTCs are found at extremely low concentrations in the blood (approximately one CTC may be

observed in 5 × 109 blood cells). While there are many techniques to recover CTCs from blood

samples, the techniques for single CTC manipulation are not sufficient, and there are no

methods designed that totally prevent loss and/or damage to some CTCs. Terstappen et al.

demonstrated a method of single-CTC manipulation using fluorescence-activated cell sorting

(FACS) after CTC recovery with the CellSearch system, which is the gold standard clinical

platform; however, only 20% of the total CTCs could be isolated at the single-cell level from

recovered samples.16 Recent studies have reported novel technologies using microfluidic

devices for single CTC isolation.17-19 Although these technologies have demonstrated a more

efficient method of single CTC isolation, the procedure only works as a low-throughput method.

For example, DEPArray (Silicon Biosystems, Bologna, Italy), currently the only commercial

platform for CTC manipulation, achieved semi-automated single-cell manipulation with high

accuracy; however, the technique takes 10 minutes to isolate single-cells, and results in a 40%

loss of input samples with a large dead volume.20-21 Therefore, a high-throughput technology for


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the manipulation of single-cells with minimal loss is required for more advanced analysis of

CTCs.

We developed a system for cancer cell recovery from blood using a microcavity

array (MCA),22-24 and have proposed a novel cell manipulation method through the visualization

of single cells through hydrogel encapsulation, referred to as “gel-based cell manipulation”

(GCM) for subsequent genetic analysis of single CTCs.25 Highly efficient recovery of CTCs

was demonstrated using the MCA system, determined based on differences in cell size and

deformability. A recent clinical study showed the practicability of the MCA system for the

enumeration of CTCs in metastatic lung cancer patients.26-27 Furthermore, CTCs isolated using

MCA were manipulated by our newly developed technique—GCM—which includes the

addition of a photopolymerized hydrogel—polyethylene glycol diacrylate (PEGDA)—into the

cells entrapped on the MCA, followed by the encapsulation of each CTC by projecting

excitation wavelengths of light using confocal laser-scanning microscopy.25 The encapsulated

single CTCs could be visualized by the naked eye and could be easily handled with tweezers

without the need for microscopic observation. We demonstrated the simple manipulation of

single CTCs recovered from blood samples without loss of cells, as well as the feasibility of



single-cell whole genome amplification (WGA). However, GCM has a throughput limitation

due to the following issues: 1) the photopolymerization of hydrogel-encapsulated single-cells

requires each cell to be individually irradiated by the confocal laser-scanning microscopy, and

2) a total of 3 min light irradiation is required for the photopolymerization of each cell in the

PEGDA hydrogel.

In this study, we developed a multiple single-cell encapsulation system for the

high-throughput manipulation of CTCs by integrating a wide-field fluorescence imaging system

and digital micromirror device (DMD). Because the wide-field imaging system can be used for

accurate and rapid CTC detection with one-shot imaging, the multiple light-irradiation system

was developed based on it.28 The DMD consisted of an array of approximately one million

micromirrors, and has been widely used as a digital light-processing (DLP)-based projector.29

Recent studies have showed the utility of DMD for stereo-lithography without the need for a

mask.30-33 By integrating these technologies, the multiple light-irradiation system resulted in a

200-fold increase in the throughput of our hydrogel encapsulation by parallel control of light

irradiation. Furthermore, we demonstrated single-cell labeling using different geometric shapes

of hydrogel to distinguish individual cells and to verify the utility of single-cell gene mutation


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analysis.

Experimental

Cell culture and staining

The non-small cell lung cancer (NSCLC) cell lines NCI-H1975 (ATCC CRL5908)

and A549 (ATCC CCL-185) were cultured in RPMI-1640 medium containing 2 mM

L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10% (v/v) fetal bovine serum (FBS;

Invitrogen, Carlsbad, CA, USA) and 1% (v/v) penicillin/streptomycin (Invitrogen) for 3–4 days

at 37 °C with 5% CO2. Immediately before each experiment, confluent cells were trypsinized

and re-suspended in phosphate-buffered saline (PBS). Cells were stained with 5 µM CellTracker

Green 5-Chloromethylfluorescein Diacetate (CMFDA; Thermo Fisher Scientific, Waltham, MA,

USA)

or

5

µM

CellTracker

Orange

5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Thermo Fisher

Scientific) for 30 min and centrifuged at 400 × g for 3 min to obtain cell pellets. After washing

twice with PBS, the cells were re-suspended in PBS containing 2 mM EDTA and 0.5% bovine

serum albumin (BSA; PBS-EB).



Cell entrapment on the MCA

The MCA, made from nickel, was fabricated by electroforming as described

previously.22 The fabricated MCA had 3,969 (63 × 63) circular pores with a diameter of 8 µm

and a distance of 125 µm between each pore. We previously confirmed this MCA could

completely recover the NCI-H1975 cells (100 cells) spiked in 1 ml blood at flow rate of 100

µl/min.25 The single-cell entrapment device equipped to the MCA was fabricated as described

previously.22 The vacuum microchannel was connected to a peristaltic pump. Cancer cells in

PBS-EB (1 ml) were introduced into the single-cell entrapment device. A negative pressure was

applied to the sample using a peristaltic pump. The sample was then passed through the MCA at

a flow rate of 100 µl/min. To wash the entrapped single cells, PBS-EB (1 ml) was passed

through the MCA at a flow rate of 100 µl/min for 10 min. In the spike-in experiment,

CellTracker Orange-stained NCI-H1975 cells were spiked in 1 ml peripheral blood

mononuclear cells (PBMC). PBMC was isolated from human healthy blood (purchased from

Tennessee Blood Services) using Histopaque®-1077 (Sigma Aldrich). For importing process,

blood was stored for 60h at 4°C. Cancer cell-spiked PBMC was processed as described above

and entrapped cells were stained by 5 µM SYTO9 (Thermo Fisher Scientific, Waltham, MA,


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USA). To remove excess dye, PBS-EB (1 ml) was passed through the MCA at a flow rate of

100 µl/min for 10 min.

Construction of the multiple light-irradiation system

A schematic diagram of the multiple light-irradiation system developed in this study

is shown in Fig. 1. The developed system contained the following basic components: mercury

vapor short arc lamp (U-HGLGPS; Olympus, Tokyo, Japan) (Fig. 1A), two projection optics for

fluorescence imaging and multiple light-irradiation, tapered light pipe homogenizing rods

(#63-103, Edmund Optics) (Fig. 1B), a UV cut filter (#62-974; Edmund Optics, Barrington, NJ,

USA) (Fig. 1C), a dichroic long pass filter I (#69-876; Edmund Optics) (Fig. 1D), excitation

filter (described in detail below) (Fig. 1E), beam splitter (#48-913; Edmund Optics) (Fig. 1F), a

dichroic long pass filter II (#69-875, Edmund Optics) (Fig. 1G), 4× objective lens (UPLFN4×;

Olympus) (Fig. 1H), emission filter (described in detail below) (Fig. 1I), DMD (DLP9000; 2560

× 1600 pixels with a pixel size of 7.6 × 7.6 µm2; Texas Instruments, Dallas, TX, USA) (Fig. 1J),

and a complementary metal-oxide semiconductor (CMOS) sensor (PL-D729MU; 3840 × 2484

pixels with a pixel size of 2.4 × 2.4 µm2; PixeLINK, Ottawa, Canada) (Fig. 1K).



This system enabled 2D fluorescence imaging in an area of 9.2 × 6.0 mm2, and

irradiation by DMD-modulated light across an area of 6.0 × 6.0 mm2 at a resolution of 7.6 × 7.6

µm2 without the scanning component. As shown as Fig. 1C, the dichroic longpass filter I split

the collimated light into two wavelengths: excitation light (λ > 430 nm) and curing light (λ =

400–430 nm). The excitation light passed through excitation filter (Fig. 1E), beam splitter (Fig.

1F), dichroic longpass filter II (Fig. 1G), and objective lens (Fig. 1H) to illuminate the entire

area of the MCA. The fluorescence signals of the target cells were focused on the CMOS sensor

after passing through two objective lenses, dichroic longpass filter II, the beam splitter, and the

emission filter (Fig. 1I). The curing light then illuminated the DMD chip (Fig. 1J). Light

modulated by the DMD was projected onto an MCA after passing through two objective lenses

and a dichroic longpass filter. All lenses used in this proposed system were selected using

OpticStudio optical simulation software (Zemax, Kirkland, WA, USA). For fluorescence

imaging, the following filter combinations were used: bandpass excitation filters (Ex): 464.5–

499.5 nm (FF01-482/35-25; Semrock, New York, NY, USA), bandpass emission filters (Em):

517.5–532.5 nm (FF01-525/15-25; Semrock) for the detection of CellTracker Green and

SYTO9; and Ex: 511–551 nm (FF01-531/40-25; Semrock), Em: 573–613 nm (FF01-593/40-25;


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Semrock) for detection of CellTracker Orange. Fluorescence images were acquired by the

CMOS sensor controlled by IC Capture 2.2 imaging software. The DMD (DLP9000; 2560 ×

1600 pixels with pixel size of 7.6 × 7.6 µm2; Texas Instruments, Dallas, TX, USA) was

controlled by LightCrafter 9000 (Texas Instruments).

Hydrogel encapsulation of single cells using the multiple light-irradiation system

The procedure for hydrogel encapsulation of single cells on the MCA using the

multiple light-irradiation system is shown in Fig. 2. Once single cells were entrapped on the

MCA, a PEGDA prepolymer containing 0.1% benzophenone (Tokyo Chemical Industry, Tokyo,

Japan) and 0.1% 4,4′-Bis(dimethylamino)benzophenone (Tokyo Chemical Industry) as the

photoinitiator were introduced onto the single-cell entrapment device. A coverslip (24 × 40 mm)

was then mounted on the single-cell entrapment device. The single-cell entrapment device was

then subjected to the multiple light-irradiation system. In this study, we used PEGDA (number

average molecular weight [Mn] = 700 Da; Sigma Aldrich) for single-cell isolation, as we

identified in our previous report that it is ideal for single-cell isolation.25

Fluorescence images of entrapped single cells were captured using the CMOS sensor



with the appropriate exposure time and were recorded as RGB color bitmap images. Candidate

cells were selected by image segmentation in each channel. Segmentation was performed based

on the fluorescence intensity and size of the objectives. Final selection of cells was made by the

operator. Subsequently, the shape of light for single-cell irradiation was assigned for each cell,

and the final irradiation pattern was generated as a binary bitmap image. Image analysis and

irradiation pattern generation were performed using custom software programmed in C#. The

light irradiation pattern was loaded to the DMD, and the entrapped single cells were exposed to

light (16.7 mW/cm2) with a wavelength of 400-430 nm through the DMD for 30 s (total light

dose: 0.5 J/cm2). PEGDA hydrogels were fabricated by free radical polymerization. The

photopolymerized PEGDA hydrogels encapsulating single cells were collected by peeling the

coverslip from the MCA. Hydrogels were washed in PBS-EB to remove excess PEGDA

prepolymer and single-cell encapsulation was confirmed by observation using fluorescence

microscopy (BX53; Olympus). Finally, each hydrogel was transferred to a 200-µl PCR tube

using tweezers for subsequent genetic analysis. For easy manipulation, all hydrogels were

designed with a width of 150 µm.


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Scanning electron microscopy (SEM)

First, the PEGDA hydrogels were fixed with 2% glutaraldehyde and 2%

paraformaldehyde in 30 mM HEPES buffer (pH 7.4) for 60 min at 4 °C. Subsequently, the

hydrogels were dehydrated with 30 mM HEPES buffer and a graded ethanol series of 25%, 50%,

75%, 90%, and 100%. The 100% ethanol was replaced with 1,1,1,3,3,3-hexamethyldisilazane,

incubated for 5 min, and air dried. Finally, the hydrogels were coated with gold by sputtering

using the E-1010 ion sputter (Hitachi, Ltd., Tokyo, Japan) and observed using SEM (VE-9800;

Keyence Corp., Osaka, Japan). The width of hydrogels were measured using ImageJ software.34

WGA of hydrogel-encapsulated single cells

Single cells encapsulated on the hydrogels were subjected to WGA using the

PicoPLEX WGA Kit (New England Biolabs, Ipswich, MA, USA), according to the

manufacturer’s protocol. Briefly, twelve cycles of pre-amplification steps and fourteen cycles of

amplification steps were performed. WGA products were purified using the MinElute PCR

Purification kit (Qiagen, Hilden, Germany). The final DNA concentration was determined on a

e-Spect spectrometer (ES-2; Malcom Co., Ltd., Tokyo, Japan).



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PCR and DNA sequencing for genotyping

PCR of WGA products was carried out at a final volume of 50 µl containing 1 µl

WGA product as a template, 0.3 µM of each primer, and 1.25 U TaKaRa Ex Taq polymerase

(Takara Bio, Shiga, Japan). All PCRs were performed using the following conditions: 2 min at

98 °C, followed by 35 cycles of 10 s at 94 °C, 2 min at 56 °C, with a final annealing step of 2

min at 72 °C. The following primers were used for the detection of mutations in exon 20 of the

epidermal

growth

factor

receptor

(EGFR)

gene;

EGFR-Exon20-T790M:

forward:

5′-CTCCCTCCAGGAAGCCTACGTGAT-3′, reverse: 5′-TTTGCGATCTGCACACACCA-3′.

PCR products were purified using the QIAquick PCR Purification kit, and their lengths were

analyzed using the Agilent DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA). PCR

products were further sequenced on the ABI PRISM 3130 DNA Sequencer (Applied Biosystems,

Foster City, CA, USA) to confirm the mutations in the isolated cells.

Results

Microscopic analysis of hydrogels photopolymerized by the multiple light-irradiation system


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Prior to single-cell encapsulation, the morphology of the PEGDA hydrogels

generated by the DMD was investigated. A square pattern (200 x 200 µm2) of light (at 800 µm

intervals) was projected onto a coverslip with hydrogel precursors using the multiple

light-irradiation system (Fig. 3A). After removing uncured hydrogels, the surface of the

coverslip was observed under a bright-field microscope. As shown in Fig. 3B, square-shaped

hydrogels were generated in the corresponding positions. Furthermore, SEM imaging revealed

the successful generation of square-shaped hydrogels (Fig. 3C). The length of the sides of the

hydrogels was 155 ± 0.4 µm, smaller than that of the irradiated light pattern (200 µm). This

phenomenon could have been due to the shrinking of hydrogel precursors during solidification.

To evaluate the performance of the proposed system, various shapes of hydrogels

were also generated (Fig. 4). Columnar hydrogels with different cross-sections [cylindrical (230

µm in diameter), square (225 µm each side), triangular (350 µm each side), cross (340 µm x 340

µm), and hollow square (225 µm each side)] were successfully fabricated (Fig. 4B). The width

of the hydrogels was found to be as designed and within ±9% of the relative error (n = 1 for

each shape), and their designed geometric features were reflected in the light irradiation patterns

(Table S1). Fig. 4C is a magnified image of part of a cross-shaped hydrogel. A mesh-like pattern



was observed on the surface of the hydrogels. This pattern was reflected by the structure of each

digital micromirror (7.6 µm × 7.6 µm).

Cell imaging by one-shot capture and multiple cell encapsulation by the DMD

To perform single-cell encapsulation using the, NCI-H1975 cells spiked in 1 ml of

PBS-EB were trapped on the MCA . NCI-H1975 cells were stained with CellTracker Green.

The surface of the MCA was then filled with PEGDA and covered with a coverslip. The cells

were visualized within the image area of the MCA using one-shot image capture (Fig. 5A).

Based on the images, light irradiation patterns were generated by a laboratory-made program

and illuminated onto the entrapped cells (Fig. 5B). After removing the uncured hydrogel, the

polymerized hydrogels were observed under a microscope. It should be noted that the

polymerized hydrogels were attached to the coverslip, but not to the MCA (Fig. 2(VI)).

Bright-field images indicated that hydrogels were polymerized in the corresponding positions

(Fig. 5C). Unexpectedly, the faces of the hydrogels were rectangular, rather than square. Further

work is required to elucidate the reasons for this. Furthermore, fluorescence imaging revealed

the successful encapsulation of a single NCI-H1975 cell, as shown in Fig. 5C. These


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experiments were repeated with different cell concentrations. Fig. 6 shows the relationship

between the number of generated hydrogels and encapsulated single cells. When all the cells on

a surface were encapsulated, the encapsulation rate was defined as 100%. The average

encapsulation rate from ten trials was 96.6 ± 5.6%. In addition, we previously demonstrated that

the MCA could capture cancer cells from 1 ml of blood at a recovery rate of 99.5% at these

concentrations.25 Therefore this result indicated that most of the cells detected as CTCs on the

MCA could be manipulated at the single-cell level. The process for multiple single-cell

encapsulation could be completed within 1 min. To further confirm the utility of multiple cell

encapsulation system, detection and multiple encapsulation of single-cancer cells recovered

from PBMC were investigated. Cancer cell and white blood cells were successfully

distinguished by fluorescence staining (Fig. S1). Average encapsulation rate from three trials

was 97 ± 2%. There are no significant difference in encapsulation rate between PBS-spiked

cancer cells and blood-spike cancer cells. This result indicated our system have a potential for

isolation of CTC from clinical sample.

Single-cell labeling using hydrogels



As shown in Fig. 4B, the shape of the hydrogels was variable based on the projected

pattern of light. In this study, we performed single-cell labeling using two different hydrogels to

distinguish between NCI-H1975 cells (with EGFR mutation (T790M)) and A549 cells (with

EGFR wild type). A mixture of stained NCI-H1975 (CellTracker Orange) and A549

(CellTracker Green) cells were analyzed using the MCA for cell entrapment, and the entire

image area of the MCA was obtained using one-shot image capture. Then, a binary image of

stained NCI-H1975 cells and A549 cells was constructed to determine the position of each cell

based on the one-shot florescence images (Fig. 7A). The square-shaped light for NCI-H1975

cells and triangular-shaped light for A549 cells were illuminated for single-cell labeling (Fig.

7B, C). The two differently shaped hydrogels containing encapsulated single-cells were

observed under bright-field microscopy (Fig. 7D). Fluorescence images of the hydrogels

revealed the successful encapsulation of each cell onto the corresponding hydrogels (Fig. 7E, F).

Furthermore, the effect of the hydrogel on WGA and genotyping of single cells was investigated.

Average DNA yields obtained by WGA were 6.0 ± 0.9 µg (n = 9) for single cells encapsulated

in square-shaped hydrogels (NCI-H1975 cells), and 4.9 ± 0.7 µg (n = 8) for single cells

encapsulated in triangular-shaped hydrogels (A549 cells). In addition, the specific sequence of


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EGFR was investigated by Sanger sequencing. A mutation in EGFR exon 20 (T790M) in

NCI-H1975 cells was successfully detected by Sanger sequencing in NCI-H1975 cells, while

the wild-type sequence was detected in A549 cells (Fig. 8). These results indicate that WGA

was successfully performed on single cells encapsulated on the hydrogel. Furthermore, to

investigate the utility of multiple labeling, we performed quadplex labeling. Two different

CellTracker-stained NCI-H1975 cells were entrapped on MCA and fluorescence intensities were

measured. CellTracker Orange-stained cells were illuminated using square-, circular-, and

diamond-shaped light. CellTracker Green-stained cells were illuminated triangular-shaped light.

In consequence, single NCI-H1975 cells were successfully isolated in each shaped-hydrogel

(Fig. S2).

Discussion This study described a multiple single-cell encapsulation system which can be used

for the high-throughput manipulation of CTCs. The multiple single-cell encapsulation system

involves a wide-field fluorescence imaging system for CTC detection,28 which enables the rapid

visualization of all stained cells in 10 sec within the field of the MCA via one-shot imaging.

This rapid detection method also allows for the rapid completion of single-CTC manipulation.



The proposed system achieved an approximately 200-fold increase in throughput for cell

encapsulation (222 cells/3 min) compared to our previous method (1 cell/3 min).25 The

encapsulated single CTCs could be visualized by the naked eye and easily handled with

tweezers without the need for microscopic observation. Although micromanipulation is

commonly used for single CTC isolation, it takes a lot of time (5–10 min per CTC).12, 35 Recent

studies have reported that the number of CTCs in 1 ml blood is at most 100 cells in late-stage

cancer patients.11,

36

For micromanipulation, it takes approximately 3–7 h to isolate 100

single-cells37; this is a time-consuming and labor-intensive process. Our GCM and multiple

single-cell encapsulation system could be applicable for high-throughput cell manipulation

without such a labor-intensive process. Besides, we previously developed a fully automated

process for the enrichment and immunostaining of CTCs.24 Therefore, through the integration of

these systems, the total time requiring human intervention was as low as 40–60 min for the

isolation of 100 CTCs; the CTC detection process takes the most time. Therefore, this multiple

single-cell encapsulation system can be utilized for high-throughput analysis of single CTCs. In

addition, we previously demonstrated that the MCA system could be used for the entrapment of

other cell types, such as microorganisms, through high-speed processing (

High-Throughput Manipulation of Circulating Tumor Cells Using a

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