Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells

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Letter

A scalable multilayer cell collector to capture circulating tumor cells with an unlimited volume capacity Yu-Lin Tsai, Po Ying Yeh, Chun-Jen Huang, Chin-Lin Guo, and Ying-Chih Chang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00315 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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ACS Biomaterials Science & Engineering

A Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells with an Unlimited Volume Capacity Yu-Lin Tsai, † Po-Ying Yeh,  Chun-Jen Huang, *, ‡ Chin-Lin Guo, *, ‖ Ying-Chih Chang *, †,  † Genomics 

Research Center, Academia Sinica, 128, Academic Road, Section 2, Nankang, Taipei 115, Taiwan

Department of Chemical Engineering, Stanford University, 443 Via Ortega, Stanford, California 94305, USA

Department of Biomedical Sciences and Engineering & Department of Chemical and Materials Engineering, National Central University, Taiwan. ‖ Institute of Physics, Academia Sinica, 128 Academic Road, Section 2, Nankang, Taipei 115, Taiwan ‡

Email: [email protected] (C-J Huang); [email protected] (C-L Guo); [email protected], [email protected] (Y-C Chang) KEYWORDS Circulating tumor cell, affinity-based cell separation, cell collector, cell-materials interaction, surface modification, antibody-PEG-coating, non-fouling, circulating system, extracorporeal apheresis ABSTRACT: Circulating tumor cells (CTCs) have been suggested as the precursors of metastatic cancer. CTC-based characterization has thus been used to monitor tumor status before the onset of metastasis and has shown to be an independent factor. The low abundance of CTCs however makes it challenging to employ CTC as a clinical routine, thus being impossible to address tumor heterogeneity. Here, we present a cell collection prototype for an efficient capture of CTCs from a large volume of body fluids such as blood. An antibody-PEG modified multilayer matrix column is engineered and connected to an apheresis-based circulation system. This setup allows us to capture CTCs repetitively from an unlimited sample volume through the circulation system, thereby increasing the capture count. Compared to conventional CTC capturing devices where the sample handling is generally limited to 1 – 10 ml, our collector is able to handle a wide range of fluidic sample (40 – 2000 ml) at a high flow rate (400 ml/min). By processing 90 minutes in circulation, we obtained average capture efficiency of at least 75 % for the colorectal cancer cell line HCT116 spiked either in 40 – 200 ml buffer solution or in a 40 ml whole blood sample. This result highlights a possibility to construct personalized CTC libraries through high-throughput CTC collection for the study of tumor heterogeneity in precision medicine.

Introduction To date, cancer has become one of the major life-threatening diseases due to its ability to metastasize. The survival rate significantly drops when cancer metastasis occurs.1-2 Monitoring the progression of cancer and preventing its metastasis has therefore become one major clinical goal. For this purpose, imaging-based methods such as contrast-enhanced computed tomography, magnetic resonance imaging, positron emission tomography, and biochemistry-based assays have been developed and used clinically for the detection and monitoring of cancer stages.3-4 These methods, however, are designed to detect tumors that have already metastasized and colonized, and hence cannot be used for a direct prevention or control of cancer metastasis. Theoretically, cancer metastasis requires cancer cells to escape from the primary site, enter the circulation system, disseminate and colonize at distant organs. It was suggested that circulating tumor cells (CTCs), i.e., cancer cells detected in the bloodstream, are the precursors of metastatic

cancers.5 In this regard, an efficient way to detect and collect CTCs before the onset of metastasis will not only help monitoring cancer progression and screening cancer cell types, but also make it possible to predict or even reduce the probability of cancer metastasis by, e.g., constructing 3D tumoroid or patient-derived xenograft (PDX) models for the study of tumor evolution, tumor heterogeneity, tumorimmunology, and drug resistance. Indeed, liquid biopsybased protocols have been established to estimate the cancer progression through the analysis of blood-isolated CTCs.6-7 CellSearchTM, for example, is a magnetic bead-based CTC isolation platform that yields zero CTC count per 7.5 ml of blood sample for all non-metastatic colorectal carcinoma (CRC) patients,1 however, with most isolated CTCs being dead or damaged due to the high shear stress in the isolation process. Our previously developed biomimetic CTC microfluidic platform, CMxTM, on the other hand, is currently one of the most sensitive CTC-detecting platforms.

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Recent results showed that high CTC counts from CMx could help monitoring cancer progression of head-and-neck carcinoma, evaluating metastatic risks of pancreatic ductal adenocarcinoma, and detecting pre-cancer of colorectal cancer.8-10 With an 80% positive detection rate for nonmetastatic CRC patients by using merely 2 mL of peripheral blood (Stages I~III, n = 95). We have shown that for nonmetastatic patients with CTC counts > 4 per 2 ml of peripheral blood, the chance to develop post-surgery metastasis within the first year is seven times higher than patients with CTC counts smaller than 5 per 2 ml of peripheral blood.11-13 These clinical results have shown that CTC quantity clearly correlates with the clinical outcome. Therefore, studying their characteristic beyond counting can elucidate the cancer biology. However, single digit CTC counts found in most cancer patients are insufficient to address tumor heterogeneity or to reconstruct a CTC profile. To increase the CTC quantity, we propose to develop a CTC capture platform that could process the widest possible range of quantity of fluidic specimen. We previously reported that using CMx the median CTC counts of stage IV and non-metastatic CRC patients are about 30 cells/2 ml, and 5 cells/2 ml, respectively.13 Hence, by increasing sample volume to 40 ml, for instance, the expected median CTCs counts may be effectively increased by 20-fold; that is, ~600 CTCs in late stage, and ~100 CTCs in early stage, as a rough estimation. Though the quantity is still small, it has met the minimal requirements for many downstream applications, including sequencing analysis to reconstruct CTC genomic profile, and increase the successful rate for cell culture and xenograft.14-15 Here, we present a Multilayer Cell Collection System consisting of a peristaltic circulatory pump, controllers, sensors, air traps and a Multilayer Cell Collector Column (MCCC) to collect CTCs from a large volume of samples with high capture efficiency. The concept is described in Scheme 1. The circulatory components were adapted from a commercially available clinical grade apheresis machine, and the MCCC is the primary component for CTC isolation, which we developed in this study.

Scheme 1. Schematic diagram of the Multilayer Cell Collection System, consisting of a Multilayer Cell Collector Column (MCCC) and a modified extracorporeal apheresis circulation system to enable large quantity of fluid process at a high volumetric flow rate.

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This setup allows us to use high processing speed to capture CTCs repetitively from the circulation system, thereby reaching a virtually unlimited sample volume to increase the captured CTC count. Given that CTCs are rare (one out of billions of blood cells) in the bloodstream, we maximize the probability that CTCs can contact the antibody coated surface and develop specific adhesion therein by rolling an antibody-PEGylated stainless-steel mesh sheet (Ab-PEG sheet, Fig. 1) into a multilayer matrix inserted in a cylindrical tube (Fig. 2). Flow-pattern controllers are implemented to increase CTCs-matrix interaction, as well as to prevent cell clogging due to the presence of large quantities of blood cells and platelets in the whole blood sample.

Figure 1. (A) The step-by-step surface modification procedures for the fabrication of the antibody conjugated sheet, “Ab-PEG sheet”. (B) Fluorescent image of the CY3-PEG sheet based on the “NA-PEG sheet”, the precursor of “Ab-PEG sheet” (scale bar: 100 um), indicating a successful large area surface modification over millimeters.

Results Fabrication of the CTC-interacting mesh sheet Fig. 1 summarized the design and fabrication steps for the AbPEG-Sheet (details can also be found in Methods and Materials): A 500 μm-thick PVC film was attached on a 500 μm-thick stainless steel mesh surface by a 50 μm-thick double-sided 3M tape, followed by CO2 laser carving to create a periodic pattern of kite-shaped PVC bumps (Fig. 1A Top). The surface of this hybrid laser carved sheet was then chemically modified to form an antibody coating. The surface modification steps are as follows: 16-17 First, O2 plasma followed by 3-aminopropyl triethoxysilane (APTES) treatment were applied to create an amine functionalized surface.18 A sequential treatments of Nhydroxysuccinimide- polyethylene glycol-biotin (NHS-PEGBiotin) and neutravidin (NA) to functionalize the surface, forming “NA-PEG sheet” (Fig. 1A, Middle), as a non-fouling underneath layer to reduce the non-specific cell-matrix


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interaction. Finally, the CTC capturing sheet, “Ab-PEG sheet”, was created by further incubating the “NA-PEG sheet” with the biotinylated antibody, anti-EpCAM (Fig. 1A, Bottom). To confirm the successful surface modification, biotinylated cyanine-3 (CY3), a green fluorescent tag, was incubated with the “NA-PEG sheet” to form a CY3-PEG sheet. As shown in Fig. 1B, the green fluorescent signal from the conjugated CY3 dye were well-distributed over the entire stainless mesh (the image shown is above millimeter scale), suggesting the NA-PEG sheet is capable of undergoing biotinylation surface reaction. The biotinylated Ab was conjugated followed by the same protocol to produce the final Ab-PEG-sheet for cell binding (Fig. 1A).

Figure 2. (A) The front view of the hybrid laser carved sheet. (B) The side view of the hybrid laser carved sheet. (C) The rolling concept and structure of the column matrix. (D) The illustration of MCCC components assembled by a multilayer matrix, outer tube, flow controller, inlet and outlet connectors. (E) The cross-sectional and (F) the lateral views of the Multilayer Cell Collector Column prototype.

Design and fabrication of the MCCC. To increase the CTC capture efficiency, we created a MCCC by adapting the concept of bioreactors, in which a large Ab-PEG-coated metallic-polymeric hybrid mesh sheet is compacted in a confined 3D space. Initially, a metallic-polymeric hybrid mesh sheet was fabricated by attaching a PVC film onto a 260 mm x 20 mm stainless-steel mesh, as shown on Fig. 2A and 2B. The film was pre-carved by CO2 laser to form periodic patterns of PVC bumps of a height of 0.45 mm (Fig. 2B), which served as interlayer spacers and defined the interlayer distance when the sheet was rolled into a multilayer matrix. The surface was modified with Ab-PEG as illustated in Fig. 1A. The distance between each bumps is 1 mm. These kite shaped patterns can split the flow stream, prolong the cell retention time and promote interlayer mixing (Fig. 2C). The stainless-steel mesh (pore size 100x100 μm) instead of flat sheet was selected to allow flow to further interpenetrate across the matrix layers. The interlayer spacing of 0.45 mm, as defined by the height of PVC bumps, was optimized to allow large flow rate yet small shear stress. 0.45 mm is much larger than typical cells (O(~10 um) in diameter) or micro-emboli (O(~10 um) to O(~100 um) in diameter). Our design ensures no change of pressure drop or clogging even if the surface was fully occupied by adhesive cells and clusters.

The matrix was then assembled with two 3D-printed flow connectors, two flow spoilers, and a plastic tube to form the column (Fig. 2D). The cross-sectional and the lateral views of the packed column MCCC were shown in Fig. 2E and 2F. Integration of MCCC and an extracorporeal apheresis circulatory system. An extracorporeal apheresis is a process involving removal of whole blood from a patient or donor to separate a certain component of whole blood, and the remaining components are re-transfused into the patient or donor. Such processes have been widely used for the kidney dialysis to separate the waste from the patient blood using reverse osmosis dialyzer, or stem cell collection using low speed centrifugation. In this study, we adapted a renal dialysis machine (Dialog+® Hemodialysis System, B Braun Medical Inc, Bethlehem USA) to collect CTCs from the sample solutions to the MCCC. The commercially available dialysis machine setup includes a peristaltic pump, a pressure sensor, an air sensor, a shutter, an air bubble trap, and the tubing supply, all of which were designed and manufactured to operate clinically. The semi-permeable membrane dialyzer is a disposable column for single use. We replaced the dialyzer with the MCCC to collect CTCs while retaining the remaining components (Scheme 1) for accurate control of flow and instant detection of abnormal pressure drop and air bubble. The shutter would close to prevent air trap in the MCCC, when the sensor is alarmed. The MCCC has been designed to be compatible with the existing specification of the existing apheresis machine. We would then operate MCCC followed by the clinically approved parameters, including the flow rate, and the corresponding shear stress. Performance of the MCCC. The flow rate of the apheresis machine used in this study can be set from 50 ml/min to 400 ml/min as a clinically safe zone for renal dialysis, as instructed by the manufacturer.19 In this report, we optimize our process based on the maximally allowed volumetric flow rate Q = 400 ml/min to maximize the sample-processing rate, as well as to evaluate the biocompatibility of MCCC under the maximal capacity. We anticipate that the parameters optimized based on the maximal flow rate would be applicable in a reduced flow rate condition. To proceed, we first applied a sample containing ~12000 pre-stained human colorectal cancer cell line HCT116 in 40 ml (V = 40 ml) phosphate buffer solution (PBS). To monitor the capture performance of MCCC over time, we collected 200 μl of fluid through a bypass to estimate the cell number remained in the sample fluid at 15 min time interval till 90 min. With these data, we can then calculate the cell ratio in fluid, P(n), defined as the fraction of the cells remained in the fluids after n times circulation through the MCCC: 𝑃(𝑛) =

𝑁(n) N(0)

(1)

where N(n) is the cell number in the fluidic sample at n times circulation. Conversely, n is in relation with the processing time t, volumetric flow rate Q, and volume of fluidic sample V, 𝑛 =

𝑄 𝑉

∙𝑡

(2)



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At Q = 400 ml/min, V = 40 ml, and t at 15, 30, 45, 60, 75 and 90 min, the corresponding lap numbers n are 150, 300, 450, 600, 750 and 900, respectively. Fig. 3A shows that the cell ratio in fluid, P(n), was exponentially decreased over circulation number n when the cell solution was circulated through MCCC (Ab-PEG-sheet, blue triangles). The cell ratio P(n) was quickly decreased to 0.35 with the lap number n at 150, then slowly reaching a plateau of 0.1 to 0.05 when lap numbers n are between 600 to 900. We used a blank control where the MCCC was coated with PEG-only to prevent nonspecific binding, but not yet coated with antibody molecules (i.e. “NA-PEG sheet”). As a result, the cell ratio in fluid P(n) over n does not follow the same exponentially decayed pattern: By the first measurement at n = 150, P value has dropped and reached a plateau at ~0.8. This number did not change over the remaining operation time period (up to 90 min, or n = 900), suggesting that the cell loss is not due to active binding by the MCCC when there is no antibody present. Instead, we found that the cell ratio change was primarily attributed by the non-uniform cell distribution in the flow system, where higher cell concentration was found in the MCCC due to the physical obstacles imposed on the flow passages, causing longer retention time in the MCCC. After the circulation was stopped and the MCCC was rinsed off with fresh PBS, we observed the recovery of cell ratio near 1.0 for the MCCC with the NA-PEG sheet, while no observable change of cell ratio for the MCCC with Ab-PEG sheet. In addition, sampling errors, counting errors, cell death, non-specific binding or cell trapped in the flow system are accounted for the systematic error ~10%. Altogether, this result confirms the feasibility of using MCCC system to actively collect CTCs as evidenced by the significant reduction of cell ratio in fluid.

Figure 3 (A) The cell ratio P of HCT116 cells in 40 ml PBS over processing circulation for the MCCC with Ab coating (Ab-PEG sheet) and the blank control (NA-PEG sheet, i.e., no Ab coating). (B) The relationship between ln P and -nm with sample volumes of 40 ml, 200 ml and 2000 ml, and linear regression based on Equation (10). R2 = 0.96, slope c = 0.003. (C) Capture efficiency 1-P versus lap number n for the samples of 40 ml, m = 1, 200 ml, m = 1, and 2000 ml, m = 6. (D) Cell ratio P of HCT116 spiked in 40 ml whole blood after 90 min (n = 900) processed by MCCC. White blood cells (WBC), red blood cells (RBC) and


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cancer cells (HCT116) were reduced by 3%, 10% and 75%, respectively. *All samples were prepared with an initial cell concentration at ~300 cells/ml, and have at least 3 repeats.

𝑃(𝑛) =

𝑁(𝑡𝑒𝑓𝑓) 𝑁(0)

= 𝑒 ―𝐴𝑡𝑒𝑓𝑓 = 𝑒 ―𝐴 𝑛 𝐿/𝜐

or

𝑙𝑛(𝑃) = ― 𝐴 𝑛 𝐿/𝜐 Processability of wide range of fluidic quantity from 10~1000 ml. The apheresis circulatory system we used in this study was designed to clinically remove toxin from whole blood of kidney failure patients. Clinically, the system is operated on an adult patient for 3-4h at the flow rate of 400 to 500 ml/min. Considering an adult has total blood volume of ~6000 ml, this operation is equivalent to ~10 to ~20 circulation numbers for each dialysis treatment.19 In other words, the dialyzer is fully capable of processing 120,000 ml blood in 4h. We attempt to evaluate the performance of MCCC for various quantities of cell solutions, in particular its compatibility for the blood samples in the order of magnitude, O(10 ~1000 ml). Though the CTC numbers widely fluctuate with patient individually, previous data has shown that the mid-range of CTC counts per ml of blood in metastatic patients is in the order of magnitude, O(1~100), with rare incidents of over 1000s; hence, we designed the total accessible surface area of each MCCC to be around 1010 um2, significantly larger than the surface area needed to form a saturated CTC layer. Even if there are over 106 CTCs, the maximal number we anticipate in a clinical condition (1000s cells in a 1000 ml blood sample), it would only occupy less than 1% of effective surface area of MCCC. Therefore, the probability of each cell captured by the MCCC is an independent event. Based on this assumption, the change of cell number N in the fluid, after an infinitesimal time interval dt can be expressed as equation (3) N(𝑡 + 𝑑𝑡) ― N(𝑡) = 𝐴𝑁(𝑡)dt

(3)

, where A represents the overall binding affinity of CTCs to the column (such as antibody affinity, surface coating, and configuration such as pattern design), which then leads to equation (4) 𝑑𝑁 dt

(4)

= ― 𝐴𝑁

Integrating the above equation (4), we obtained the change of N over time: N(𝑡𝑒𝑓𝑓) = 𝑁(0)𝑒 ―𝐴𝑡𝑒𝑓𝑓

(5)

Here, teff represents the effective time by which the cells pass through the antibody coated MCCC (i.e., time spent in the uncoated tubing and connectors is not counted for “effective time” for binding). In the MCCC, the volumetric flux υ was maintained as a constant. If the blood sample has passed through the column with the length of L for n laps, then the effective time in contact with the antibody coated column reads: 𝐿

𝑡𝑒𝑓𝑓 = 𝑛. 𝑣

(6)

P(n) is the ratio of cell number after passing through the column for n laps to the original cell number, i.e.,

(7)

(8)

This expression suggests that the cell ratio P is indeed exponentially decayed with lap number n, as observed in our experiment (Fig. 3A). Conversely, at fixed linear velocity, cell ratio P can be reduced if the column length is elongated, i.e. longer effective time between the cells and MCCC. A serial connection of multiple MCCC modules can effectively elongate the total column length L. Each MCCC has a fixed column length l of 20 mm (Fig. 2C), then a serial connection of m MCCC modules would lead to total L = 𝑙 𝑚 (𝑚𝑚) = 20 𝑚

(9)

We could simplify the dimensionless expression as follows, (10)

𝑙𝑛 𝑃 = ― 𝑐 𝑛 𝑚

The equation (10) indicates that the cell capture efficiency can be parameterized by the column number m, and the number of circulating laps n, where c is a coefficient constant, 𝑐 = 𝐴𝑙

(11)

To validate this relation, logarithm of cell ratio ln P against -nm, was plotted using the following experimental runs: HCT116 cancer cell solutions of 40 ml, and 200 ml were processed by one MCCC, and a 2000 ml solution was processed by six MCCCs in a serial connection. Remarkably, all three experimental conditions fit nicely into a universal linear curve as predicted by Equation (10), where R2 = 0.96 and the coefficient constant c = 0.003 (Fig. 3B). As the change of the cell ratio P in the solution is attributed by the active adsorption by the MCCC, the capture efficiency of the MCCC may be expressed as 1-P. 1-P versus n of the three samples is plotted in Fig. 3C. Samples of V = 40 ml and V = 200 ml processed by one MCCC module fall into the same rate of capture efficiency versus n: both reach over 60% of capture efficiency at n = 150 (Fig. 3C, dash line indicates 60%). The capture efficiency of the 40 ml sample continues to increase ~95% over n = 900 (90 min). We did not continue to process the 200 ml sample over n = 900, because it would be equivalent to 450 min of processing time, which is too long. The prolonged processing time is not desirable, as it may pose the adverse effects such as losing cell viability, increasing contamination, and blood coagulation. Instead, for a large sample, processing with serial connection of several MCCC modules could achieve the equivalent capture efficiency within the desirable time constraint. For the 2000 ml sample, we connected six MCCC modules, and demonstrated that 66% capture efficiency can be achieved with n = 12 in 60 min (Fig. 3C, red circles). Capture efficiency in blood sample. To ascertain whether the collector can be used for clinical samples, cell ratios of ~2x 104 CMFDA-labeled HCT116 cancer cells spiked in the



40 ml of peripheral blood samples were measured using the same protocol. After processing by one MCCC for 900 laps (90 min), as shown in Fig. 3D, cell ratio in fluid P was quickly reduced to 0.25, equivalent to the 75% of capture efficiency. This reduction rate is comparable with the previously reported platforms (such as 73% capture efficiency for HCT116 cells in 2 ml blood by CMx in 80 min).13, 20-21 To evaluate the non-specific binding, we measured the reduction rate of blood cells simultaneously. We found that white blood cells and red blood cells were reduced by ~3% and 10%, respectively, which were within the system errors of ~10%. These results confirm the ability of MCCC to provide a high specificity on CTC detection and capturing from whole blood samples. It is noted that no pressure drop, clogging, or blood coagulation were observed in using blood based samples. Cell viability. To prevent cell loss by the flow, we optimized our inlet/ outlet and interspace distance accordingly. At the volumetric flow rate of 400 ml/min, and the average 0.75 cm2 effective cross-sectional area for the flow through the MCCC, the corresponding flux is 133 mm/s, which is in a similar range of a typical microfluidic device for cell separation.22-25 More importantly, the maximal linear velocity, as occurring at the narrowest passage between two kite-shaped bumps, is 342 mm/s, which corresponds to the maximal shear stress of 30.5 dyne/cm2 in the entire MCCC. This maximal shear stress value is well below the shear stress value of ~60 dyne/cm2, a threshold value that may begin inducing cell damages.13, 26-27 The cell viability of the cells in the circulatory fluid and those adsorbed on the MCCC sheet were further assessed after processing by MCCC system. In this experiment, ~1 x 107 HCT116 cells were spiked in 200 ml of PBS and circulated for 90 min. Subsequently, 100 μl of circulatory fluid as well as a 5 mm x 5mm cut mesh sheet were collected and stained using cell viability kit (LIVE/DEAD® Viability/Cytotoxicity Kit, Thermo Fisher Scientific), where live cells were in green and the dead cells were in red. By counting the live cells and dead cells, we obtain the cell viability to be ~83% in the circulatory system (Fig. 4A for live cells and Fig. 4B for dead cells) and ~84% adsorbed on the MCCC cut sheet (Fig. 4C for live cells and Fig. 4D for dead cells).

Figure 4. Representative images of (A) live (Green) and (B) dead (Red) cells from the fluidic sample, in comparison with the representative images of (C) live (Green) and (D) dead (Red) cells directly stained on the MCCC sheet.

Discussion

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The design of current prototype has some distinctive features differing from conventional affinity based microfluidic design. In a microfluidics, the inner channel’s volume-to-surface ratio has to be low in order to increase the contact probability of the cells in solution with the antibody surface. Because of this inherent limitation, microfluidics is an excellent platform for small quantity of specimen, but not suitable for large quantity of specimen. In our current design, we enlarge the inter-space so that high volumetric flow rate but low shear stress is possible. We have demonstrated a process condition with operating flow rate at 400 ml/min, yet less than 30.5 dyne/cm2 shear stress, to achieve capture efficiency over 90% in buffer solution, and 75% in blood. With such a low shear stress, we minimize the risk of blood clogging or cell damage. As shown by the cell viability test, ~84% cells are viable in the fluid and on the mesh after 90 min operation at 400 ml/min, or 180 circulation, and no blood cells are significantly damaged. Our experimental results fit nicely to a universal curve, indicating that the system is scalable by which ln P is proportional to - nm, product of lap number and MCCC module number in a serial connection. We demonstrated the operation range from 40 ml to 2000 ml, but we anticipate that this could further widen to 8 ml or whole body fluidic volume. The MCCC system has shown several unique advantages. The stainless-steel mesh provides a strong mechanical support for column fabrication and operation. The multilayer bioreactor design generates a large surface area for CTC capturing within a compact column. The fabrication and assembly are straightforward. Furthermore, after completing the cell capture and purification, the multilayer rolling sheet could be quickly released from the column container and unrolled to a flat sheet. The captured cells could be observed, cultured, and manipulated directly on the sheet. This unique origami design is convenient such that no cell elution from the Collector is required. Without cell elution, we further reduce the cell loss/ damage. We demonstrated the capability of MCCC for processing wide range of fluid volume with consistent capture efficiency. There are several reasons we would like to process high quantity of fluids: First, given that most people can donate 500 ml of blood in one blood bag, once the MCCC can be applied clinically, with the 75% capture efficiency of blood samples, we can obtain and analyze CTCs potentially up to 500 ml blood sample. This means ~102 to ~106 CTCs or ~1000 to 6000 median CTC counts could be collected in 2h processing time. For most of patients, this quantity would be sufficient to generate a personalized tumor profiles and for drug testing. Secondly, our prototype is designed and built to fit in an extracorporeal apheresis machine, meaning that the MCCC can be a pilot design for a direct isolation of CTCs from the human body to reduce the concentration of CTCs in patient’s blood, which may have pending therapeutic benefits. Thirdly, previous data has shown that the blood viscosity and the high-density cell population would inevitably interfere the cell capture efficiency. For example, for a tube of 10 ml blood sample, after diluting to 40 ml, the capture efficiency was improved from 75% to 85% using MCCC. Therefore, the capture efficiency in buffer solution always outperform that in the


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whole blood. Ability to enlarge sample size by dilution could be a convenient approach to improve the capture efficiency. Conclusion. In summary, we provide a Multilayer Cell Collection System which is scalable for capturing CTCs from a wide range of sample sizes with high capture efficiency and viability. We believe that the presented technology could potentially lead to the breakthrough in CTC heterogeneity analysis and ultimately to suppress cancer metastasis. This work has enabled the future clinical studies in precision medicine. Methods and Materials Surface modification of the hybrid sheet. The hybrid laser carved sheet was oxidized with plasma for 120 seconds and then soaked into the (3-aminopropyl) triethoxysilane (APTES) solution (Sigma-Aldrich, MO), which was made by dissolving 15 uL of APTES into 1 ml of 95% alcohol.13 After incubating the mesh with APTES for 1h, the mesh was dehydrated for another 1h at 55°C, followed by 1h treatment of 0.1 ml EZ-Link™ NHS-PEG12-Biotin (Thermo Fisher Scientific) solution, which was made by dissolving 1 mg of EZ-Link™ NHS-PEG12-Biotin in 1 ml PBS. The treatment enabled the addition of neutravidin (Thermo Fisher Scientific) and biotin labeled anti-EpCAM (eBioscience, modified following the previous protocol17-18 on the mesh surface. In this work, NA solution was made by dissolving 1 mg of NA into 1 ml of PBS and the antibody was made by dissolving 0.1mg of anti-EpCAM (EpAb4-1) in 1 ml PBS. The total volume of NA or antibody used for the mesh modification was 0.1 ml. Cell line labeling and spiking. The human colorectal cancer cell line HCT116 was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) and used as EpCAM positive cell line. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 1% antibiotic-antimycotic (ThermoFisher Scientific) and 10% fetal bovine serum (FBS; Gibco) under 5% CO2 humidified atmosphere. To measure the capturing efficiency, the HCT116 cancer cells were pre-stained with 20 μm of CellTracker Green CMFDA dye (Life technologies, Carlsbad, CA) under 37°C for 20 minutes. The labeled cells were then spiked into PBS or blood from healthy donor. The blood drawn protocol was approved by the IRB-Biomedical Science Research, Academia Sinica, under AS-IRB-BM14026v3. All donors had read and signed informed consent forms. Evaluation of non-specific adsorption of CTCs in MCCC System. A cell solution was prepared by ~80 HCT116 cell line /ml spiked in total 40 ml of PBS (i.e. 3000 cells in total). The cell solution was run through the circulatory system with the MCCC column comprised of NA-PEG sheet, a blank control with no antibody coating. At 60 min of circulation, 60 cells/ml were remained in the PBS circulatory fluid, as estimated by the 100 ul bypass fluid sampling. After gently rinsing the MCCC, total ~3000 cells were fully recovered, indicating negligible cell loss during the circulatory process or non-specific adsorption at MCCC. REFERENCES 1. Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.;

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Detection. PLoS One 2016, 11 (3), e0149633. DOI: 10.1371/journal.pone.0149633. 14. Eifler, R. L.; Lind, J.; Falkenhagen, D.; Weber, V.; Fischer, M. B.; Zeillinger, R., Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: proof of concept. Cytometry B Clin Cytom 2011, 80 (2), 100-11. DOI: 10.1002/cyto.b.20560. 15. Yu, M.; Bardia, A.; Aceto, N.; Bersani, F.; Madden, M. W.; Donaldson, M. C.; Desai, R.; Zhu, H.; Comaills, V.; Zheng, Z.; Wittner, B. S.; Stojanov, P.; Brachtel, E.; Sgroi, D.; Kapur, R.; Shioda, T.; Ting, D. T.; Ramaswamy, S.; Getz, G.; Iafrate, A. J.; Benes, C.; Toner, M.; Maheswaran, S.; Haber, D. A., Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science 2014, 345 (6193), 216-20. DOI: 10.1126/science.1253533. 16. Meng, S.; Liu, Z. J.; Shen, L.; Guo, Z.; Chou, L. S. L.; Zhong, W.; Du, Q. G.; Ge, J., The effect of a layer-by-layer chitosan-heparin coating on the endothelialization and coagulation properties of a coronary stent system. Biomaterials 2009, 30 (12), 2276-2283. DOI: 10.1016/j.biomaterials.2008.12.075. 17. Lai, C. H.; Choon Lim, S.; Wu, L. C.; Wang, C. F.; Tsai, W. S.; Wu, H. C.; Chang, Y. C., Site-specific antibody modification and immobilization on a microfluidic chip to promote the capture of circulating tumor cells and microemboli. Chem Commun (Camb) 2017, 53 (29), 4152-4155. DOI: 10.1039/c7cc00247e. 18. Chen, L. M.; Zheng, L.; Lv, Y. H.; Liu, H.; Wang, G. C.; Ren, N.; Liu, D.; Wang, J. Y.; Boughton, R. I., Chemical assembly of silver nanoparticles on stainless steel for antimicrobial applications. Surface & Coatings Technology 2010, 204 (23), 3871-3875. DOI: 10.1016/j.surfcoat.2010.05.003. 19. Bhimani, J. P.; Ouseph, R.; Ward, R. A., Effect of increasing dialysate flow rate on diffusive mass transfer of urea, phosphate and beta(2)-microglobulin during clinical haemodialysis. Nephrology Dialysis Transplantation 2010, 25 (12), 3990-3995. DOI: 10.1093/ndt/gfq326. 20. Yeh, P. Y.; Chen, Y. R.; Wang, C. F.; Chang, Y. C., Promoting Multivalent Antibody-Antigen Interactions by Tethering Antibody Molecules on a PEGylated Dendrimer-Supported Lipid Bilayer. Biomacromolecules 2018, 19 (2), 426-437. DOI: 10.1021/acs.biomac.7b01515. 21. Lu, S. H.; Tsai, W. S.; Chang, Y. H.; Chou, T. Y.; Pang, S. T.; Lin, P. H.; Tsai, C. M.; Chang, Y. C., Identifying cancer origin using circulating tumor cells. Cancer Biol Ther 2016, 17 (4), 430-438. DOI: 10.1080/15384047.2016.1141839. 22. Shen, Z.; Wu, A.; Chen, X., Current detection technologies for circulating tumor cells. Chem Soc Rev 2017, 46 (8), 2038-2056. DOI: 10.1039/c6cs00803h. 23. Lagus, T. P.; Edd, J. F., J Phys D Appl Phys 2013, 46 (11). DOI: 10.1088/0022-3727/46/11/114005. 24. Hofmann, O.; Niedermann, P.; Manz, A., Modular approach to fabrication of three-dimensional microchannel systems in PDMS - application to sheath flow microchips. Lab Chip 2001, 1 (2), 108-114. DOI: 10.1039/b105110p. 25. Holmes, D.; Pettigrew, D.; Reccius, C. H.; Gwyer, J. D.; van Berkel, C.; Holloway, J.; Davies, D. E.; Morgan, H., Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry. Lab Chip 2009, 9 (20), 2881-2889. DOI: 10.1039/b910053a. 26. Regmi, S.; Fu, A.; Luo, K. Q., High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System. Sci Rep 2017, 7, 39975. DOI: 10.1038/srep39975. 27. Lu, H.; Koo, L. Y.; Wang, W. M.; Lauffenburger, D. A.; Griffith, L. G.; Jensen, K. F., Microfluidic shear devices for quantitative analysis of cell adhesion. Anal Chem 2004, 76 (18), 5257-64. DOI: 10.1021/ac049837t.

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This work was supported by grants from the Academia Sinica, and Ministry of Science and Technology under contract MOST106-2218-E-001-004 and MOST107-2119-M-001-039. Chin-Lin Guo acknowledges the support from MOST104-2112M-001-043-MY3, MOST106-2627-M-001-005, MOST1062918-I-001-005, MOST107-2112-M-001-040-MY3, and MOST107-2119-M-001-039, and fund from Academia Sinica, AS-105-TP-A04. Anti-EpCAM (EpAb4-1) was provided by HanChung Wu, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

ACKNOWLEDGMENT


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For Table of Contents Use Only A Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells with an Unlimited Volume Capacity Yu-Lin Tsai, Po-Ying Yeh, Chun-Jen Huang, Chin-Lin Guo, Ying-Chih Chang.



Scheme 1. Schematic diagram of the Multilayer Cell Collection System, consisting of a Multilayer Cell Collector Column (MCCC) and a modified extracorporeal apheresis circulation system to enable large quantity of fluid process at a high volumetric flow rate. 148x84mm (300 x 300 DPI)

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Figure 1. (A) The step-by-step surface modification procedures for the fabrication of the antibody conjugated sheet, “Ab-PEG sheet”. (B) Fluorescent image of the CY3-PEG sheet based on the “NA-PEG sheet”, the precursor of “Ab-PEG sheet” (scale bar: 100 um), indicating a successful large area surface modification over millimeters. 228x191mm (300 x 300 DPI)



Figure 2. (A) The front view of the hybrid laser carved sheet. (B) The side view of the hybrid laser carved sheet. (C) The rolling concept and structure of the column matrix. (D) The illustration of MCCC components assembled by a multilayer matrix, outer tube, flow controller, inlet and outlet connectors. (E) The crosssectional and (F) the lateral views of the Multilayer Cell Collector Column prototype, and the top views of the inlet and outlet spoilers. 168x189mm (300 x 300 DPI)


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Figure 3 (A) The cell ratio P of HCT116 cells in 40 ml PBS over processing circulation for the MCCC with Ab coating (Ab-PEG sheet) and the blank control (NA-PEG sheet, i.e., no Ab coating). (B) The relationship between ln P and -nm with sample volumes of 40 ml, 200 ml and 2000 ml, and linear regression based on Equation [1]. R2 = 0.96, slope c = 0.003. (C) Capture efficiency 1-P versus lap number n for the samples of 40 ml, m = 1, 200 ml, m = 1, and 2000 ml, m = 6. (D) Cell ratio P of HCT116 spiked in 40 ml whole blood after 90 min (n = 900) processed by MCCC. White blood cells (WBC), red blood cells (RBC) and cancer cells (HCT116) were reduced by 3%, 10% and 75%, respectively. *All samples were prepared with an initial cell concentration at ~300 cells/ml, and have at least 3 repeats. 46x117mm (300 x 300 DPI)



Figure 4. Representative images of (A) live (Green) and (B) dead (Red) cells from the fluidic sample, in comparison with the representative images of (C) live (Green) and (D) dead (Red) cells directly stained on the MCCC sheet. 109x81mm (300 x 300 DPI)


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Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells

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