Remote Control in Formation of 3D Multicellular Assemblies Using

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Tissue Engineering and Regenerative Medicine

Remote control in formation of 3D multicellular assemblies using magnetic forces Javad Jafari, Xiao-lian Han, Jason Palmer, Phong A Tran, and Andrea Janet O'Connor ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00297 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 14, 2019

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ACS Biomaterials Science & Engineering

Remote control in formation of 3D multicellular assemblies using magnetic forces Javad Jafari†, Xiao-lian Han‡, Jason Palmer‡, Phong Tran†,§ and Andrea J O'Connor†,* Department of Biomedical Engineering, Particulate Fluids Processing Centre, The University of Melbourne, Grattan St, Parkville, Victoria 3010, Australia †

O'Brien Institute Department, St Vincent’s Institute, 42 Fitzroy Street, Fitzroy, Victoria 3065, Australia ‡

Interface Science and Material Engineering Group, School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, 2 George St, Brisbane Queensland, 4000, Australia §

Corresponding Author: * Email: [email protected]

Abstract Cell constructs have been utilized as building blocks in tissue engineering to closely mimic the natural tissue and also overcome some of the limitations caused by two dimensional cultures or using scaffolds. External forces can be used to enhance the cells` adhesion and interaction and thus provide better control over production of these structures compared to methods like cell seeding and migration. In this paper, we demonstrate an efficient method to generate uniform, three dimensional cell constructs using magnetic forces. This method produced spheroids with higher densities and more symmetrical structures than the commonly used centrifugation method for production of cell spheroids. It was also shown that shape of the cell constructs could be changed readily by using different patterns of magnetic field. The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications. Keywords: Tissue Engineering, Magnetic field, Magnetic particles, Spheroids, 3D culture, Coculture 1 ACS Paragon Plus Environment

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1. Introduction Engineering soft tissues, and specially adipose tissue, for healing defects caused by injury or trauma is a very important aspect of tissue engineering. These problems may affect the quality of patients` lives cosmetically or mentally other than the functional impairment 1-2. However, challenges can arise in the application of scaffolds in tissue engineering due to issues including inflammatory reactions caused by biomaterials or their degradation products and also foreign body reactions

3-6.

Also, while monolayer or 2D culture is extensively used in cell

research, it does not adequately replicate the complexity of tissue microenvironments; it suffers from inadequate cell-cell and cell-extra cellular matrix (ECM) interactions and signaling that are vital in natural, in vivo tissues 7-8. The alternative of animal studies is expensive, ethically more difficult and may not match cell and tissue behaviour in humans. By mimicking 3D in vivo structures, cell aggregates and specifically spheroids can be used widely in drug screening 9-10 or play role as building blocks in tissue engineering 11-16 and help to fill in the gap between animal models and 2D culture 8. Traditional methods of making spheroids, as an important 3D model in tissue engineering 17, include using microfluidics 18-20, polymer matrices 21-22, hanging drop methods 23-26 and centrifugation 13, 27-28. However, there are some limitations associated with these methods such as difficulties in changing the culture media, limited volume of the cell suspensions, overall costs of using special plates and also difficulties in scaling up the methods 7. Also, while external force could influence the behaviour of cells during and after formation of spheroids as mechanical stimuli, methods like hanging drop lack an active force to provide sufficient contact between cells and fusion of aggregates 25.

This, in turn, can limit the spatial control of the cells and also delay the formation process. Magnetic fields provide a potential method for indirect application of forces onto

magnetically labelled cells and biomaterials, with excellent spatial and temporal control. For example, magnetic fields can be used to create a driving force for generation of cell spheroids 2 ACS Paragon Plus Environment

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11, 15.

By putting magnets above cell culture plates, cells labelled with magnetic particles could

be levitated and agglomerated under the culture medium meniscus to form spheroids

29-32.

However, this process usually requires a long assembly time, even when the field is concentrated to enhance the spheroid formation 33. Also, such methods do not offer easy control of the shape of the cell constructs 34. In this paper magnetic fields have been utilised in various configurations to drive the formation of different shapes and sizes of 3D cellular aggregates. Smaller aggregates can be combined through remote control to create larger constructs using magnetic fields. The forces on the cells and constructs can be controlled simply via spatial control of the magnetic field in which they reside and the forces can be applied, maintained, or removed as desired during and after construct production. 3T3-L1 pre-adipocytes have been used as potential precursor cells for adipose tissue engineering and the formation of multicomponent constructs patterned in three dimensions with fibroblast cells is demonstrated.

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2. Materials and methods 2.1.

Cells and culture

NIH 3T3 fibroblasts and 3T3-L1 pre-adipocytes (ATCC® CL173™) were cultured at 37 °C under a humidified atmosphere of 5% CO2 and 95% air in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, NSW, Australia) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Life Technologies, Australia), L-Glutamine (0.5%) (Sigma-Aldrich, Australia), and streptomycin/penicillin (0.5%) (Sigma-Aldrich, Australia). The culture media was changed every 3 days.

2.2.

Labeling the cells with magnetic particles

For labeling the both cell types with magnetic particles (MPs), they were first cultured in cell culture flasks until ~90% confluence. Then, fresh culture media containing 30 μg/mL of sterilized ~4.5 μm carboxylated MPs (Spherotech Inc, Australia) were added followed by incubation for 24 h to allow the cells to attach to the particles. Details on the optimization of the particle concentration and incubation time are given in the Supporting Information (S1, S2). While degradable nanoparticles are used in some studies

35,

relatively large MPs were

chosen here to reduce the possibility of their internalization by pre-adipocytes in order to avoid potential unintended effects of endocytosed particles and to enable mechanotransduction across the cell membranes (Supporting Information S3). After washing the cells with phosphate buffered saline (PBS), twice to remove the unattached particles, trypsin/EDTA (SigmaAldrich, Australia) was used to detach the cells from the culture surface. Magnetic separation was utilized to separate labeled from unlabeled cells for 3 minutes. The required concentration of cells for culture was determined with the aid of a haemocytometer.


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2.3.

Formation of the cell constructs

Production of cell aggregates was performed by utilizing low adhesion surfaces. To accomplish this, 96 well plates were coated with

poly (2-hydroxyethyl methacrylate)

(PolyHEMA) (Santa Cruz Biotechnology, Thermofisher Scientific) according to standard protocols (Supporting Information S4) 36. To examine the formation of different shapes of cell constructs, both flat- and round-bottom 96 well plates were used. An array of cylindrical neodymium magnets (M21: d= 2 mm, h=1 mm; MicroWings, Queensland, Australia) was used to produce the dominant MF under the culture plates (Supporting Information S5). For the next step, 2.0×104 pre-adipocyte cells labeled with MPs were added to the wells in the presence and absence of magnets. The plates were shaken by hand in a figure of 8 pattern for 2 minutes and then incubated at 37 °C. Production of constructs was studied every 2 h by inverted and bright field microscopy (Olympus BX51).

2.4.

Simulation of magnetic fields

To simulate the magnetic field generated by M21 cylindrical magnets under flat- and round-bottom 96 well plates and study its effect on formation of different shapes of cell aggregates, Finite Element Method Magnetics (FEMM) software (https://www.femm.info) was employed. This software provides a two-dimensional heat map of the magnetic flux density according to the distance from the surface pole 33. The thickness of the flat and roundbottom plates at their base was 0.5 and 1.14 mm, respectively according to their manufacturer (Costar).

2.5.

Production of spheroids by different methods

The assembly of cell spheroids using magnets and the efficacy of the magnetic field (MF) in establishing cell-cell contact was compared to using centrifugation which is a typical means of fabricating spheroids. In this regard, 2.0×104 pre-adipocyte cells, un-labeled and 5 ACS Paragon Plus Environment


labeled with MPs were cultured in low adhesion, round-bottom 96 well plates and then centrifuged at 1000g for 10 minutes (called: Cent-MPs, Cent+MPs for unlabelled and labelled cells, respectively) 27. In another group, labeled cells, in the absence of centrifugation, were exposed to the MF for 10 minutes before removing the M21 magnets for the rest of the experiments (called: Short MF+MPs) contrary to the main group in which the magnets stayed in place for the whole experiment time (10h) (called: Long MF+MPs). For the control group, un-labeled cells were cultured without the introduction of any magnetic particles or field force. Images were captured using an inverted microscope (Olympus IX71, Australia) after 10 h and then, size and roundness (inverse of aspect ratio) of the spheroids were calculated by using imageJ software (https://imagej.net).

2.6.

Merging the cell spheroids

The feasibility of making larger and more complicated structures was evaluated. To do so, two or four spheroids were transferred into a single well and imaged by inverted microscopy. After incubation at 37 °C for 24 h in the presence of a M21 magnet under the plate, optical images were captured again and samples prepared for SEM. Another pair of spheroids were stained separately with green and red CellTracking dyes (Abcam, VIC, Australia) and after following the same protocol, they were examined by fluorescence microscopy. To perform the patterning of cell spheroids into a rod and a ring assembly, ring magnets (R211: douter=2 mm, dinner=1 mm, h=1 mm; MicroWings, Queensland, Australia) were positioned under the plate, and five or ten spheroids were added into the wells, respectively. The structures generated were imaged by inverted bright field microscopy at different time intervals up to 96 h.


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2.7.

Core-Shell cell spheroids

To produce a hybrid structure from two types of cells and also to investigate the effect of magnetic fields on assembling uniform core-shell constructs, first, pre-adipocyte spheroids were placed inside low adhesive 96-well plates in the presence (+MF) and absence (-MF) of M21 magnets. Then, 2.0×104 3T3 fibroblasts labeled with MPs and stained with green CellTracking dye were added to each well and incubated overnight. The formation and consistency of cell layers were examined by optical and confocal laser scanning microscopy (Leica TCSPC confocal microscope, Australia).

2.8.

Cell morphology

To compare the morphology of the cells and also the cytoskeleton distribution in 2D and 3D culture, 2.0×104 pre-adipocyte cells labeled with MPs were cultured on 15 mm glass coverslips coated with 50 µg/mL fibronectin (Life Technologies Australia Pty Ltd) overnight. This monolayer and also pre-adipocyte cell spheroids prepared as above were washed twice with PBS and fixed in 4% (w/v) paraformaldehyde for one hour. They were then permeabilized by 0.5% (w/v) Triton® X-100 (Sigma-Aldrich, Australia) in PBS and their F-actin and nuclei were stained with 5 µg/mL Alexa Fluor 488 phalloidin (Sigma-Aldrich, Australia) and 0.1 µg/mL DAPI (Sigma-Aldrich, Australia) for 20and 10 min, respectively. Finally, cultures were imaged by confocal laser scanning microscope.

2.9.

Scanning Electron Microscopy

For scanning electron microscopy (SEM), cell constructs were fixed with 4% (w/v) paraformaldehyde for one hour and washed twice with PBS. Afterwards, they were dehydrated with serial dilutions of ethanol (20, 50, 70, 90, and 100% (v/v) in water) for 10 minutes each and then dried using hexamethyldisilazane under a fume hood overnight. Finally, samples were



gold sputter coated using a Dynavac SC100 (Dynavac Engineering, Australia) and images were captured using FEI Quanta Cryo SEM.

2.10.

Histology

For histological analysis, spheroids were fixed in 4% paraformaldehyde (w/v) for one hour and then mounted in 1.5% (w/v) agarose solution (90% distilled water and 10% formalin) for 10 minutes. After placing the solid agarose block in 4% (w/v) paraformaldehyde for 60 minutes to stabilize, it was washed with PBS and placed in biopsy cassette and processed on a 24-hour cycle (Excelsior tissue processor, Thermo Scientific, USA) and embedded in paraffin. Samples were then sectioned at 5 µm thickness and mounted on glass slides. The staining of cell nuclei and cytoplasm was performed by using haematoxylin and eosin according to a standard protocol 37 for 5 and 2 minutes, respectively. For apoptosis studies, cleaved caspase3 labeling was performed using an immuno-peroxidase method involving primary antibody, biotinylated secondary antibody (Vector, Calif., USA), ABC complex (Vector) and DAB chromogen (Dako, Calif., USA). Isotype controls were performed in order to confirm staining specificity. More details are outlined in reference 38.

2.11.

Live/dead cell evaluation

To perform live/dead staining after 2 and 10 days, cell spheroids were washed with PBS twice and Acridin Orange (AO, 0.5 µg/mL, Sigma) and Propidium Iodide (PI, 0.5 µg/mL, Sigma) were used to stain the live (green) and dead (red) cells, respectively. After adding AO/PI working solution (1:1 volume ratio) for 30 min, samples were rinsed twice with medium and imaged by confocal laser scanning microscopy. A composite z-stack of 36 images with intervals of 1 µm was obtained from the surface of each spheroid and then percentage of live and dead cells was quantified from the z-projection of images using ImageJ software.


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2.12.

Statistical analysis:

Statistical data analysis was performed by a two-tailed Student`s t-test. The data is reported as mean ± standard deviation and significance was considered at p 24 h) were needed, even using a higher magnetic flux density ~0.065 T (compared to ~0.051 T used here)

31.

Increasing the magnetic flux density up to 7 times (to ~0.45 T)

33

reduced the assembly time somewhat to 18 h. Formation of cell spheroids with MPs by methods such as hanging drop and centrifuge rather than using magnetic driving forces also requires at least 24 h 24, 28, 41-42. Using magnets placed below the cell culture plates, as shown here, allows gravitational and magnetic forces to work together, enabling assembly to occur with lower magnetic flux densities, enhancing cell-cell contacts and reducing the assembly times.



Figure 3. Formation of 3T3-L1 cell spheroids in the presence (+MF) and absence (-MF) of an applied magnetic field after different time points of culture (scale bars: 300 µm)

Figure 4. Diameter of 3T3-L1 cell spheroids in presence of magnetic field (+MF) in different time points after culture (n=4)

Appearance of the cell constructs in both plates was checked by bright field microscopy and SEM. Bright field microscopy presented orange color images from spheroids due to the presence of MPs in the assemblies. The shapes of the culture wells affected the shapes of the structures produced, with more spherical structures produced in the round-bottom wells and disc-shaped structures in the flat-bottomed wells (Figure 5). This demonstrates that the changes in magnetic field acting on the cells with the different well shapes (shown by the finite element simulations) were sufficient to result in distinctly different assemblies. It was also evident in higher magnification of SEM images that cells secreted ECM which covered the surface of the aggregates and MPs (Figure 5E).


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Figure 5. Bright field and SEM images of 3T3-L1 cell constructs produced in round- (A, C) and flat-bottom (B, D) plates using magnetic particles and fields (scale bar: A, B, C= 200 µm; D= 300 µm; E=20 µm); The color of the aggregates in bright field images is related to presence of magnetic particles; Extra cellular matrix produced by cells has covered the surface of the spheroid (E); the white arrow points to one of the magnetic particles appearing covered under the ECM.

3.3.

Production of spheroids by different methods

As shown above, arranging the magnetic field to align the magnetic and gravitational forces on the cells by positioning magnets under the cultures plates enhances the formation of pre-adipocyte spheroids, making the assembly process much faster than previously reported. This may be due to enhanced cell-cell interactions in this configuration, one of the main steps in assembling 3D cell constructs

43-44.

We compared the potential of magnetic force-based

aggregation to centrifugation. Figure 6A shows that the introduction of MPs to the cells led to 15 ACS Paragon Plus Environment


a significant increase in size of the spheroids produced by centrifugation using the same number of cells. Exposure of cells to MF for 10 min. (Short MF+MPs) produced similar sized cell spheroids (910±30 µm) to 10 min. of centrifugation (Cent+MPs). However, using MF for 10 h (Long MF+MPs) resulted in assembly of significantly smaller constructs (830±20 µm). It was evident that exposure to MF for 10 h had provided more efficient force compared to the centrifuge method in the experimental time period and enhanced cell-cell contact leading to formation of more compact and consistently sized structures. This was also evident by comparing the size of spheroids in Long MF+MPs and Short MF+MPs samples. When size of the spheroids in Cent-MPs was compared to other groups, this difference could be explained by two reasons. First, in the samples with MPs, cell-cell interactions were limited by the presence of MPs which then reduced the contraction of cell structures. Also, addition of MPs` volume to the volume of cells can be considered as the other factor which increased the overall volume of the spheroid compared to the sample without MPs. However, it seems that the former had more effect; otherwise there should not be a significant difference in the size of the structures between Cent+MPs and Short MF+MPs groups with Long MF+MPs samples. The roundness of the aggregates also tended to be more consistent when the MF exposure was longer (Figure 6B). As expected, culturing the cells in the absence of a centrifugal or magnetic force did not result in a single uniform aggregate per well (data not shown).


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Figure 6. (A) Mean diameters of spheroids produced from 3T3-L1 cells in different situations; Cent-MPs: unlabeled cells and centrifuge for 10 min, Cent+MPs: labeled cells and centrifuge for 10 min, Short MF+MPs: 10 minutes exposure of labeled cells to the magnetic field, and Long MF+MPs: exposure of labeled cells to the magnetic field for 10 hours; (B) Roundness of spheroids produced from 3T3-L1 cells in different situations; Cent-MPs: unlabeled cells and centrifuge, Cent+MPs: labeled cells and centrifuge, Short MF+MPs: 10 minutes exposure of labeled cells to the magnetic field, and Long MF+MPs: exposure of labeled cells to the magnetic field for 10 hours (n=8; * p

Remote Control in Formation of 3D Multicellular Assemblies Using

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