Development of a Magnetic 3D Spheroid Platform with Potential

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Development of a magnetic 3D spheroid platform with potential application for high-throughput drug screening Wei Mei Guo, Xian Jun Loh, Ern Yu Tan, Say Chye Joachim Loo, and Vincent H.B. Ho Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5000604 • Publication Date (Web): 19 May 2014 Downloaded from http://pubs.acs.org on May 26, 2014

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Molecular Pharmaceutics

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Development of a magnetic 3D spheroid platform with potential application for

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high-throughput drug screening

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Wei Mei Guoa, Xian Jun Lohb, Ern Yu Tanc, Joachim S.C. Lood, Vincent H.B. Hoa,*

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a

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138673, Singapore

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b

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Singapore 117602, Singapore

Molecular Engineering Laboratory, A*STAR, Proteos, 61 Biopolis Drive, Singapore

Institute of Materials Research and Engineering, A*STAR, 3 Research Link,

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c

11

Singapore 308433, Singapore

12

d

13

50 Nanyang Avenue, Singapore 639798, Singapore

Department of General Surgery, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng,

School of Materials Science and Engineering, Nanyang Technological University,

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* E-mail: [email protected]

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ACS Paragon Plus Environment

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Abstract

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Three dimensional (3D) cell culture has become increasingly adopted as a more

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accurate model of the complex in vivo microenvironment compared to conventional

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two dimensional (2D) cell culture. Multicellular spheroids are important 3D cell

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culture models widely used in biological studies and drug screening. To facilitate

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simple spheroid manipulation, magnetic spheroids were generated from magnetically

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labeled cells using a scaffold-free approach. This method is applicable to a variety of

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cell types. The spheroids generated can be targeted and immobilized using magnetic

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field gradients, allowing media change or dilution to be performed with minimal

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disruption to the spheroids. Cells in magnetic spheroids showed good viability and

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displayed typical 3D morphology. Using this platform, a 28 day study was carried out

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using doxorubicin on magnetic MCF-7 spheroids. The results provided a proof-of-

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principle for using magnetic tumor spheroids in therapeutic studies. They can offer

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beneficial insights which help to bridge the gap between in vitro and in vivo models.

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Furthermore, this platform can be adapted for high-throughput screening in drug

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discovery.

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Keywords: multicellular spheroids; magnetic cell labeling; spheroid manipulation;

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therapeutic studies; high-throughput screening


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1. Introduction

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In recent years, there has been increasing emphasis on three dimensional (3D)

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cell culture systems. This is due to the increase in demand for more physiologically-

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relevant in vitro tissue models which can recapitulate actual in vivo conditions.(1-3)

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These models provide more clinically relevant results in preclinical studies.(4, 5) Two

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dimensional (2D) cell cultures have been commonly employed for evaluating efficacy

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or toxicity of drug candidates in screening studies. However, studies have shown that

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there are significant differences in the phenotypic and functional characteristics when

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cells are grown in monolayers or 3D cultures.(6-8) The 2D environment poorly

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reflects the complex 3D in vivo microenvironment where cells are in close contact

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with other cell types and the extracellular matrix, and are subjected to cellular

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interactions that influence cell differentiation, proliferation and migration.(9, 10)

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Multicellular spheroids (MCS) are simple and widely used 3D cell culture system.

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They are usually formed by spontaneous aggregation and fusion of cells without any

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external artificial scaffolds.(11-13) Homotypic MCS can be obtained from a broad

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range of cell types(14). Different cell types can also be co-cultured to create

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heterotypic MCS.(15, 16) These microtissues exhibit high similarities to actual tissues

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in features such as cellular heterogeneity, nutrient and oxygen gradients, matrix

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deposition and cell-cell signaling.(17) MCS also offer the opportunity for prolonged

21

studies as they can be cultured for weeks in contrast to cells grown in monolayers

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which will be over-confluent in a matter of days.(18, 19)

23 24

Various approaches have been established to generate 3D spheroids. Conventional

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methods include spinner flask or liquid overlay culture(20, 21) while others have

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utilized protein(22) or synthetic polymer scaffolds.(23, 24) There are more advanced

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spheroid formation methods, which include micron-scaled substrates(25) and

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microfluidic devices.(26, 27) Although these offer certain advantages such as higher

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throughput of spheroid generation or simplified liquid handing, some of these systems

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are complicated and require sophisticated equipment and specialized training.

31 32

The hanging drop culture allows precise control over spheroid size and uses readily

33

available laboratory substrates.(14) Recently, it was shown that spheroids in hanging

34

drops could be magnetically manipulated by introducing magnetic labels into the


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spheroids.(16) This methodology was then applied in tissue engineering and drug

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screening. However, it was recognized that the hanging drop method might not be

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compatible with automated handling for higher throughput applications without

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further substrate modification.(28, 29)

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In this study, a platform consisting of magnetic microtissues for drug screening is

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developed. It provides a simple methodology for manipulating 3D spheroids on a

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larger scale.(30) Various human cell lines were magnetically labeled using a

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previously described method. Labeled cells were then seeded into low attachment 96-

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well micro plates for spheroid generation.(31) The magnetic spheroids cultured from

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labeled cells could be manipulated with magnetic field gradients. This facilitates

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complete media change with minimal disruption to the spheroids and reduces

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spheroid loss. Imaging of the microtissues can be carried out using conventional

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microscopy or automated cell imager. A Food and Drug Administration (FDA)-

15

approved drug, doxorubicin, was used as a model drug for screening studies using this

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platform.

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2. Experimental Section

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2.1 Materials

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Biotinamidohexanoic acid N-hydroxysuccinimide ester (BiotinSE, Sigma B2643),

21

366 mM in DMSO, was stored at –20°C. Streptavidin MagneSpheres paramagnetic

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particles (Promega Z5482), 1 mg mL-1, were stored at 4°C. Dulbecco’s Phosphate

23

Buffered Saline (PBS, PAA H15-002) was stored at room temperature. p-nitrophenyl

24

phosphate disodium salt hexahydrate (Sigma N9389) was stored at –20°C.

25 26

2.2 Cell culture

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The following human cell lines were used in this study: MCF-7 breast

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adenocarcinoma (ATCC HTB-22), MDA-MB-231 breast adenocarcinoma (ATCC

29

HTB-26), HepG2 hepatocellular liver carcinoma (ATCC HB-8065), A549 lung

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adenocarcinoma epithelial (ATCC CCL-185), HeLa cervical carcinoma (ATCC CCL-

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2), MCF 10A mammary epithelial cells (ATCC CRL-10317), HEK 293 human

32

embryonic kidney cells (ATCC CRL-1573), BEAS-2B bronchial epithelial cells

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(ATCC CRL-9609), BJ skin fibroblast cells (ATCC CRL-2522) were grown in

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Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco 11965) supplemented with


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10% fetal bovine serum, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin.

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Human mesenchymal stem cells (Lonza PT-2501) were cultured in Minimum

3

Essential Medium (MEM) Alpha (Gibco 12561) and HCT-116 colorectal carcinoma

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(ATCC CCL-247) in McCoy’s 5A medium (Biochrom AG F1015) with the same

5

medium supplements. H1 embryonic stem cell (WiCell Research Institute) was

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maintained in mTeSR-1 medium (Stem Cell Technologies). All cells were cultured in

7

a 5% CO2 humidified atmosphere at 37°C.

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2.3 Spheroid culture

10

MCF-7 cells were magnetically labeled using a previously established method.(32,

11

33) Briefly, the cells were treated with 750 µM BiotinSE in PBS at room temperature

12

for 30 min. Biotinylated cells were added to 0.025 mg mL-1 streptavidin paramagnetic

13

particles (0.5 – 1.5µm in diameter) and vortexed at room temperature for 15 s to

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ensure uniform mixing of cells and particles. To generate magnetic MCF-7 spheroids,

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the magnetically labeled cells were seeded at 1 × 103 in 100 µL culture medium per

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well of 96-well round bottom low attachment plate (Corning Inc. 7007). The cells

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were counted using an improved Neubauer haemocytometer. The spheroids were

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cultured in a 5% CO2 humidified atmosphere at 37°C for 3 days before drug

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treatment.

20 21

2.4 Drug treatment

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The diameter size of the spheroid used for 3D studies was ~400 µm after an initiation

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interval of 3 days when 1 × 103 MCF-7 cells were seeded per well. The magnetic

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spheroids were immobilized using a 96-Well Magnetic Separator (Invitrogen

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CS15096) and the cell culture media was removed. The magnetic spheroids were

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treated with or without doxorubicin at various concentrations: 0.01, 0.1, 1, 10, 100 µg

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mL-1, prepared in culture medium, for 24 h. After incubation at 37°C, the drugs were

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removed and the spheroids were washed with PBS. Thereafter, fresh media was added

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before further culture of up to 28 days with media refreshed every 2 days.

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2.5 Confocal microscopy

32 33

MCF-7 spheroids were stained with 20 µM fluorescein dicacetate and 25 µg mL-1

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propidium iodide for 2 h at 37°C. They were then placed on 35 mm glass-bottom


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culture dishes (MatTek) and imaged using a Zeiss LSM 5 DUO confocal laser

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scanning microscope using 10× magnification. Both dyes were excited using the 488

3

nm laser line and emission was detected at 543 nm. Z-stacks were taken until the

4

fluorescence was not detected. These images were then compiled into 3D projections

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using Zeiss LSM Image Browser Software.

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2.6 F-actin staining

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MCF-7 cells and spheroids were fixed in 4% paraformaldehyde (ChemCruz™ SC-

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281692) for 30 min and permeabilized with 0.5% Triton X-100 (Promega H5142)

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diluted in PBS for 15 min. F-actin was stained with Rhodamin phalloidin (Invitrogen

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R415) (15 µL methanolic stock solution diluted in 600 µL 0.1% BSA in PBS) for 20

12

min. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen H3570) for 10

13

min. All staining procedures were carried out at room temperature.

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2.7 Growth monitoring

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The size of the spheroids was measured by taking images of the live spheroid using an

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inverted phase contrast microscope (Zeiss AXIOVERT 40 CFL). Spheroid size was

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determined from measurements of two orthogonal diameters from which the

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geometric mean diameter was calculated.(18)

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2.8 Histology

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MCF-7 spheroids were fixed with 10% neutral buffered formalin for 2 h at 4°C and

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washed with PBS. The fixed spheroids were prepared using a VIP tissue processor

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(Sakura, Japan). The process consisted of dehydration in a series of increasing ethanol

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concentrations up to 100% and then two changes of xylene and four changes of

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paraffin wax (Paraplast) and eventual embedding in paraffin. The duration for each

27

step was 10 min. Spheroid sections were cut at 5 µm thickness and stained with

28

haematoxylin and eosin. Stained sections were imaged using a Nikon AZ 100

29

microscope.

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2.9 Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)

32 33

Total RNA was isolated from MCF-7 spheroids using Trizol® Reagent (Invitrogen

34

15596-028) with Purelink™ RNA Mini Kit (Invitrogen 12183-018A). 200 ng of total


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RNA was reverse transcribed into complementary DNA (cDNA) using Superscript™

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III First-strand synthesis system for qRT-PCR (Invitrogen 18080-051). qRT-PCR was

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performed using the StepOnePlus™ Real-Time PCR Systems (Applied Biosystems)

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and the amplifications were done using Maxima SYBR Green/ROX qPCR master mix

5

(2×) (Fermentas, K0221). The thermal cycling conditions were composed of initial

6

denaturation step at 95°C for 5 min, followed by 45 cycles at 95°C for 10 s, 57°C for

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15 s and 60°C for 50 s. The experiments were carried out in triplicate for each data

8

point and a standard curve was included. Data was normalized to that of 2D MCF-7

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cell monolayer. GAPDH was used as the housekeeping gene. The sequences for the

10

primers are:

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VEGF-A

12 13 14

5′-CTG CTG TCT TGG GTG CAT TGG-3′ (Forward) 5'-TCA CCG CCT CGG CTT GTC-3' (Reverse)

GAPDH

5′-AAGGTGAAGGTCGGAGTCAA-3′ (Forward) 5′-GAAGATGGTGATGGGATTTC-3′ (Reverse)

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2.10 Acid phosphatase assay

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Using the 96-well Magnetic Separator, the spheroids were directed to the side of the

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well, the supernatant were carefully removed and replaced with 100 µL PBS. 100 µL

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of the assay buffer (0.1M sodium acetate, 0.1% TritonX-100, supplemented with 2 mg

20

mL-1 p-nitrophenyl phosphate, Sigma N9389) was added to each well and incubated

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for 90 min at 37°C. Following incubation, 10 µL of 1N sodium hydroxide (NaOH)

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was added, and absorption at 405 nm was measured using a Tecan Infinite 200

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microplate reader.(34)

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3. Results and discussion

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3.1 Generation and manipulation of magnetic multicellular spheroids

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To generate magnetic multicellular spheroids, single cells were first labeled with

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paramagnetic particles and then seeded into round-bottomed wells of low attachment

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96-well microplates. By gravitational force, the labeled cells formed a multicellular

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aggregate within 24 h at the bottom of the well, and eventually a more compact

31

spheroid after 48 h (Figure 1A).

32 33

The method of cell labeling is based on specific affinity binding of biotin to

34

streptavidin. It is a two-step procedure, which involves biotinylation of cell membrane


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proteins, followed by attachment of streptavidin paramagnetic particles onto the

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surface of the biotinylated cells.

3 4

In previous studies (15, 35-38), cells were labeled by cellular incorporation of

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magnetic materials. Uptake quantity was unknown and the process would generally

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require an incubation period of up to 24 h, to allow cells to endocytose the magnetic

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particles. The required length of time is dependent on cellular uptake property and

8

hence may vary between different cell types. For example, the incubation periods for

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magnetic labeling of stromal fibroblast NIH3T3 and HepG2 cells with magnetite

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cationic liposomes (MCLs) were 4 h and 24 h, respectively (39). Therefore, the step-

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wise affinity binding provides a quick way to magnetically label the cells prior to

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spheroid generation.

13 14

The average diameter of the streptavidin paramagnetic particles used in this study was

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1.0 ± 0.5 µm. Studies have shown that larger paramagnetic material (> 1µm diameter)

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does not affect the intracellular signaling and cell viability as compared to sub-micron

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magnetic materials (15, 40, 41). It has been shown previously from transmission

18

electron microscopy data that residual paramagnetic particles were found within the

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spheroid after 3 weeks of culture. It was possible that intracellular uptake of the

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paramagnetic particles had occurred via either phagolysosomes or smaller endosomal

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compartments(18).

22 23

The ease of manipulation of the magnetic spheroids was illustrated in Figure 1B. The

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microplate was placed on top of a 96-Well Magnetic Separator (Invitrogen CS15096),

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consisting of an array of 24 neodymium magnets. The magnets held the magnetic

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spheroids in place at one end of the well while cell culture media was completely

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removed and exchanged for fresh medium or addition of drugs. There was minimal

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disturbance to the immobilized spheroid during pipetting, and hence, a lower risk of

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spheroid loss.

30 31

As a demonstration of the adaptive application of this method, various human cell

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lines were magnetically labeled and successfully generated into magnetic

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multicellular spheroids (Figure 2). Magnetic spheroids formed from non-tumorigenic

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cell lines could potentially be used as microtissue models in cellular or biochemical


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assays to screen for toxicity of drugs, recombinant proteins and gene therapies, or for

2

analysis of cellular microenvironment on gene expression, proliferation and apoptosis

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(12, 36). Magnetic tumor spheroids have been known to be useful cancer models in

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drug delivery research(42). The magnetic property of these spheroids enabled rapid

5

separation from drugs after incubation or washing procedures.

6 7

3.2 Structural characteristics of magnetic multicellular spheroids

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F-actin cytoskeletal organization in 2D monolayer MCF-7 cells and magnetic MCF-7

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spheroids were observed using confocal microscopy by Rhodamin phalloidin staining.

10

MCF-7 cells grown on 2D monolayer exhibited a flattened shape, with presence of

11

filopodia and lamellipodia stretching out from edges of the cells (Figure 3A). Such

12

formations were not visible in cells cultured in 3D. In contrast, the spheroid displayed

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a cortical F-actin network typical of 3D cell morphology, having close contact with

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each other at the cellular junctions (Figure 3B). Such a network is indicative of

15

cytoskeletal reorganization, when suspended cells self-assembled into 3D spheroid in

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a microgravity environment (43). The use of such culture model would be useful for

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drug screening studies, providing valuable information on the biological response of

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cells grown in 3D, which closely resembled actual in vivo tissue morphology (38).

19 20

Cell viability in the magnetic spheroids was visualized using a live/dead stain in

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confocal microscopy. Viable cells were stained green due to the conversion of

22

fluorescein diacetate to fluorescein by intercellular esterases, while non-viable cells

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that lost membrane integrity were stained red by propidium iodide. The majority of

24

magnetic labeled MCF-7 cells within the 7-day-old magnetic spheroid were viable

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(Figure 3C). This suggested that the paramagnetic particles did not seem to have

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adverse effect on cell viability and spheroid formation.

27 28

During spheroid growth, cell viability within the spheroids may be affected by the

29

lack of vascular network in the 3D microenvironment. As a result, larger multicellular

30

spheroids developed central secondary necrosis and hypoxia (20, 44), due to the

31

establishment of a diffusion gradient that would limit nutrient and oxygen delivery to

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cells at the center of the spheroids. This phenomenon was observed in the 14-day-old

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spheroid (Figure 3D), where there was a significant increase in the number of non-

34

viable cells in the central regions.


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Histological analysis of hematoxylin and eosin (H&E) stained sections of the

3

magnetic MCF-7 spheroids revealed individual cells arranged in a densely packed

4

morphology (Figure 3E). It was also shown that cells were aggregated together with

5

paramagnetic particles within the spheroid with very few dead cells observed.

6

Spheroids cultured for a much longer time were observed to have a more concentric

7

cellular arrangement with non-viable cells located in the core (Figure 3F). This

8

observation was consistent with the live/dead staining results (Figure 3D) and

9

previously reported data where mass transport limitations created a hypoxic core

10

similar to that of solid tumors(45). Notably, the paramagnetic particles remained

11

present within the older spheroid (18).

12 13

3.3 Gene expression profiles of 3D multicellular spheroids

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The expression of vascular endothelial growth factor (VEGF-A), a classic marker for

15

hypoxic stress(46-48) was investigated (Figure 4). Quantitative PCR was performed

16

using RNA from 2D and 3D cell samples. Spheroids cultured for four days had

17

VEGF-A expression patterns similar to those from 2D monolayers, which were used

18

as a baseline for non-hypoxic culture. Relative expression of VEGF-A was up-

19

regulated in spheroids cultured for nine days and became significantly elevated by day

20

14 of prolonged cultures. These data showed that necrosis observed in the H&E

21

histology staining could be induced by hypoxia (Figure 3F).

22 23

3.4 Establishment of 3D spheroid-based assay for therapeutic screening

24

The 96-well microplate format was employed in this study (Figure 5) to develop a

25

platform for standardized spheroid culturing, rapid imaging and analysis that is

26

amenable to large-scale drug screening and compatible with existing high-throughput

27

screening systems. In addition, the ease of culture medium exchange using magnetic

28

manipulation is promising for long-term spheroid culture. Culture media change could

29

be performed with ease and repeatedly on the same plate with minimal spheroid

30

disruption. The low attachment micro plates provided non-adherent surfaces that

31

prevent cell adhesion. The round bottom well shape promoted the formation of

32

reproducible and homogeneously sized spheroids. A water reservoir was constructed

33

around the periphery of the microplate to prevent a commonly encountered problem

34

where medium near the edges are more prone to evaporation.(31)


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Measurement of the growth kinetics of spheroids in response to drug treatment can be

3

carried out using conventional microscopy or automated cell imaging. In this study, a

4

benchtop in situ cellular analysis system – Celigo™ cytometer (Brooks Life Science

5

System) was used. This cytometer scans a 96-well plate within ten minutes. Bright

6

field images were acquired and analyzed. Growth curves of the multicellular

7

spheroids can be rapidly and easily generated from the multi-parametric data analysis,

8

which includes measurements of spheroid diameter, perimeter and area. Together, this

9

platform provides the opportunity for continuous observation and quantitative

10

analysis of spheroid culture in prolonged therapeutic studies with durations

11

comparable to animal studies.

12 13

3.5 Drug treatment of 3D multicellular spheroids

14

Doxorubicin, a conventional anticancer drug, was used as a model drug for

15

therapeutic studies using 3D magnetic spheroids (49, 50). Magnetic MCF-7 tumor

16

spheroids were treated with free doxorubicin over a 24-hour period before further

17

culture for 28 days. The average diameter of the spheroids at the onset of drug

18

treatment was 400 µm, with clear pathophysiological gradients and with no central

19

necrosis. Culture medium was exchanged every 48 hours using the method described

20

above.

21 22

Spheroid size was monitored over 28 days to track recovery of the spheroids. The

23

orthogonal diameters of each spheroid were measured to determine its volume. In

24

clinical setting, treatment effectiveness is usually benchmarked by a reduction in

25

tumor size. Diameter measurements to assess spheroid growth have been widely used

26

as an index of 3D proliferation (9, 20, 31). Hence, the extent of reduction in spheroid

27

volume could be indicative of drug efficacy.

28 29

The effect of drug treatment on spheroid integrity and volume was analyzed (Figure

30

6A). MCF-7 spheroids showed a dose-dependent response to doxorubicin. Growth

31

inhibition of spheroids and evident cell shedding occurred with drug treatment at 1 µg

32

mL-1 and above. For drug treatments at 0.1 µg mL-1 and below, recovery from growth

33

inhibition was observed 14 days after drug administration. This could be due to the

34

presence of diffusion gradients within the spheroids, which resulted in a reduction in


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drug penetration (20, 44) for lower drug concentrations. In addition, frequent

2

exchange of fresh culture media may have resulted in drug dilution, thus lowering the

3

effective drug concentration. This could also help to maintain nutrient supply and

4

promote the recovery of spheroid growth after the initial decrease in size.

5 6

An acid phosphatase assay was performed to analyze spheroid viability at the study

7

endpoint (21, 34). The assay result showed a similar dose-dependent effect, in which

8

an increasing doxorubicin concentration was correlated with a corresponding decline

9

in spheroid viability (Figure 6B). These results demonstrated that magnetic 3D tumor

10

spheroids could potentially be used for testing drug efficacy.

11 12

4. Conclusion

13

In this study, magnetic multicellular spheroids were generated from magnetically

14

labeled cells using 96-well round bottom low attachment plate. The methodology can

15

be applied to a wide variety of cell types for spheroid generation. Morphological

16

analysis of magnetic spheroids revealed good cell viability and structural

17

characteristics commonly observed in 3D tissue structures. The magnetic labeling of

18

cells provides a facile method to manipulate 3D spheroids with transiently applied

19

magnetic fields. This is convenient for medium exchange and can support long-term

20

cell culture. The platform developed provides a simple and versatile method of

21

screening therapeutic candidates against tumor spheroids, and is particularly valuable

22

when drug efficacy is evaluated in prolonged preclinical studies. Moreover, it has the

23

potential to be further developed for high-throughput systems.

24 25

Acknowledgements

26

The authors would like to thank both Singapore Biomedical Research Council,

27

Agency for Science, Technology and Research (BMRC, A*STAR) and National

28

Medical Research Council, Ministry of Health (NMRC/CIRG/1342/2012, MOH) for

29

funding support. Histology images were acquired in the SBIC-Nikon Imaging Centre

30

at Biopolis, Singapore.

31 32

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1

Page 18 of 26

Figure captions

2 3

Figure 1. Schematic of generation and manipulation of magnetic spheroids in

4

microplate format.

5

Cells are first labeled with paramagnetic particles and seeded in 96-well low

6

attachment round bottom plates. Spheroids formed can be directed to the side of the

7

wells by an external magnet. Upon spheroid immobilization, complete media change

8

or addition of drug can be carried out for long term culture or drug treatment studies.

9 10

Figure 2. Homotypic magnetic spheroids generated from different cell lines.

11

Formation of magnetic spheroids from non-cancer cell lines (A-F) and cancer cell

12

lines (G-L) in low attachment round bottom 96-well plates. (A) Human embryonic

13

stem cell (H1), (B) Human mammary epithelial cells (MCF10A), (C) Human

14

embryonic kidney cells (HEK 293), (D) Human bronchial epithelial cells (BEAS-2B),

15

(E) Human mesenchymal stem cells (hMSCs), (F) Human skin fibroblast cells (BJ),

16

(G)

17

adenocarcinoma (MCF-7), (I) Human colorectal carcinoma (HCT-116), (J) Human

18

hepatocellular liver carcinoma (HepG2), (K) Human lung adenocarcinoma epithelial

19

(A549), (L) Human cervical carcinoma (HeLa). Scale bars represent 200 µm.

Human

breast

adenocarcinoma

(MDA-MB-231),

(H)

Human

breast

20 21

Figure 3. Structural and morphological characterization of magnetic spheroids.

22

F-actin visualization in (A) MCF-7 monolayer and (B) MCF-7 spheroid. Cell viability

23

assay using fluorescein diacetate for staining viable cells and propidium iodide for

24

non-viable cells. (C) 7 day-old spheroid (20X magnification). (D) 14 day-old spheroid

25

(10X magnification). Hematoxylin–and-eosin staining of cross-sections of MCF-

26

7spheroids. (E) 7 day-old (F) 14 day-old. White arrows indicate paramagnetic

27

particles.

28 29

Figure 4. Relative expression of vascular endothelial growth factor (VEGF-A) in

30

magnetic spheroids.

31

The expression levels of VEGF-A in 3D MCF-7 spheroid are shown relative to the

32

expression level of VEGF-A in 2D MCF-7 monolayer cells.

33 34

Figure 5. 3D magnetic spheroid-based assay for therapeutic screening


18

Page 19 of 26

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1

Magnetic spheroids generated in 96-well plate were imaged using conventional

2

microscopy or automated cell imager to capture spheroid integrity and diameter

3

following drug treatment. Data analyses include growth curves and frequency

4

distribution curves. Assessment of drug effects on spheroids was determined through

5

the acid phosphatase assay(34) to measure spheroid viability.

6 7

Figure 6. Effect of doxorubicin on volume growth and spheroid viability in 3D

8

magnetic spheroid assay.

9

MCF-7 spheroids were treated with various concentration of doxorubicin for 24 h.

10

The drug was removed and spheroids were further cultured for 28 days. Spheroid

11

volume growth was monitored over the entire duration and spheroid viability was

12

measured at the end of the study. Values are means ± SD (n=5).

13 14


19


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Page 20 of 26

1 2

A

Cell seeding for spheroid generation

48 - 72h

B

Media change and drug addition via magnetic spheroid capture

Removal

Addi on

Magnet

3 4

Magnetic labeled single cells

Spheroids

Drug

Figure 1

5 6


20

Page 21 of 26

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1 2

A

B

C

D

E

F

G

H

I

J

K

L

Figure 2


21


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

Page 22 of 26

1

A

monolayer

spheroid

B

y x y z

C

7 day-old

D

14 day-old

E

7 day-old

F

14 day-old

100µm

2 3

Figure 3


22

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


Rela ve VEGF Expression

Page 23 of 26

1 2

Figure 4


23


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Page 24 of 26

1

Microscope with camera or Automated imager

Single spheroid per well (96-well plate)

Imaging & Analysis

• Drug treatment • Media change • Track growth

Endpoint viability assay

2 3

Figure 5


24

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1 2

3 4

Figure 6

5 6 7 8


25


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1 2 3

4 5

TOC graphic


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Development of a Magnetic 3D Spheroid Platform with Potential

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