Biofabrication

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Scaffold-free, label-free and nozzle-free biofabrication technology using

magnetic levitational assembly
To cite this article before publication: Vladislav A Parfenov et al 2018 Biofabrication in press https://doi.org/10.1088/1758-5090/aac900

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Page 1 of 28 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1

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1 Scaffold-free, label-free and nozzle-free biofabrication
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5 2 technology using magnetic levitational assembly
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10 4 Vladislav A. Parfenov1*, Elizaveta V. Koudan1*, Elena A. Bulanova1, Pavel A. Karalkin1,
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12 5 Frederico DAS Pereira1, Nikita E. Norkin1, Alisa D. Knyazeva1, Anna A. Gryadunova1, Oleg F.
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6 Petrov2, Mikhail M. Vasiliev2, Maxim I. Myasnikov2, Valery P. Chernikov3, Vladimir A.

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15 7 Kasyanov4, Artem Yu. Marchenkov5, Kenn Brakke6, Yusef D. Khesuani1, Utkan Demirci7,
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17 8 Vladimir A. Mironov1
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22 11 1
Laboratory for Biotechnological Research “3D Bioprinting Solutions”, Moscow, Russia;
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24 12 Joint Institute for High Temperatures, Russian Academy of Sciences, Moscow, Russia;
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Institute of Human Morphology, Russian Academy of Science, Moscow, Russia;
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Riga Stradins University, Riga, Latvia;
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National Research University “Moscow Power Engineering Institute”, Moscow, Russia;
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Susquehanna University, Selinsgrove, Pennsylvania 17870, USA
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32 17 Stanford University, Palo Alto, USA
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35 19
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37 20 Сorresponding authors: Vladislav A. Parfenov, Russian Federation, Moscow 115409, Kashirskoye shosse,
38 21 68-2, tel. +7 (499) 769-50-18, vapar@mail.ru; Vladimir A Mironov, Russian Federation, Moscow 115409,
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40 22 Kashirskoye shosse, 68-2, tel. +7 (499) 769-50-18, vladimir.mironov54@gmail.com; Utkan Demirci,
41 23 Stanford University, 1-650-9069227, utkan@stanford.edu
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43 24
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25 * Both authors contributed equally to this paper
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48 27 Author contributions: V.A.P., U.D. and V.A.M. designed research; V.A.P., E.V.K., F.D.P., V.P.C., A.D.K.
49 28 and A.A.G. performed research; O.F.P., M.M.V. and M.I.M. performed MD simulation of dynamics of the
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51 29
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polystyrene particles in the cusp magnetic trap; KB made surface evolver simulation, V.A.P. performed
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30 computer simulation of magnetic field; V.A.P., E.V.K., P.A.K., A.Y.M., N.E.N. and V.A.K. analyzed data;
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54 31 V.A.P., E.V.K., E.A.B., Y.D.K., U.D. and V.A.M. wrote the paper.
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2 1
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4 2 Abstract
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3 Tissue spheroids have been proposed as building blocks in 3D biofabrication.
7 4 Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling
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9 5 by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational
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11 6 assembly has been introduced. Here we present first-time report on rapid assembly of 3D tissue
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7 construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids.
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14 8 Chondrospheres of standard size, shape and capable to fusion have been biofabricated
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16 9 from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation
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18 10 was performed using a prototype device equipped with permanent magnets in presence of
19 11 gadolinium (Gd3+) in culture media, which enables magnetic levitation.
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21 12 Mathematical modeling and computer simulations were used for prediction of magnetic
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23 13 field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used
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14 polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal
26 15 settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of
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chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in

30 17 the presence of Gd3+.

31 18 Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising
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33 19 approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-
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35 20 driven tissue engineering.
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38 22 Keywords: tissue spheroids, magnetic levitation (MagLev), scaffold-free, label-free,
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40 23 chondrospheres, gadolinium (Gd3+), biofabrication.
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1
2 1 Introduction
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4 2 Tissue spheroids represent densely packed aggregates of living cells [1]. At first, they
5
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3 were used to design the human diseases models in vitro and test lead candidates at preclinical
7 4 stages of drug development [2-5]. More recently, tissue spheroids were proposed as building
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9 5 blocks for bioprinting of functional human tissues and organs [6-8].
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11 6 The research on tissue spheroids was focused on three main directions: 1) the
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7 development of robotic scalable standardized methods for tissue spheroids biofabrication [9]; 2)
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14 8 systematic phenotyping of tissue spheroids [10] and 3) the elaboration of methods for assembling
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16 9 tissue spheroids into 3D tissues and organ structures [11-13].
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18 10 Several beneficial advantages for using tissue spheroids as building blocks exist. First,
19 11 tissue spheroids have a highest theoretically possible cell density comparable with natural tissue
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21 12 [2]. Second, tissue spheroids have a compact rounded shape, which is ideally suitable for their
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23 13 handling, manipulation, transfer, processing and bioprinting. Third, they have a complex internal
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14 structure and multicellular composition. Moreover, tissue spheroids can have one or several
26 15 lumens and even can be pre-vascularized [14]. Finally, they have intrinsic properties for fusion.
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Tissue fusion is an ubiquitous phenomenon during embryonic development [15] and it is a

30 17 fundamental principle of rapidly emerging bioprinting and biofabrication technologies based on

31 18 the use of tissue spheroids as building blocks. Closely placed and directly touching each other,
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33 19 tissue spheroids begin to fuse thus producing complex 3D tissue constructs [6].
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35 20 Several approaches exist for tissue and organ biofabrication using tissue spheroids. Using
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21 one method, cell aggregates were placed robotically or manually into 3D scaffolds made from
38 22 various biodegradable biomaterials [16-18]. Another approach involves the attachment and
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40 23 spreading of tissue spheroids on electrospun matrices [19-21]. Furthermore, magnetic forces
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42 24 have been used for 2D patterning of tissue spheroids, biofabricated from cells, labelled with
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25 magnetic nanoparticles [22-26]. All described biofabrication approaches are scaffold-based,

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45 26 nozzle-based or label-based. These methods have certain advantages and inherent limitations.
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47 27 For example, robotic 3D bioprinting allows to biofabricate complex 3D tissues and organs [13,
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49 28 27, 28]. Further, the majority of current approaches involves the use of biomaterials or
50 29 nanomaterials as scaffolds. The main goal of our work was to demonstrate a path-breaking rapid
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52 30 approach for biofabrication of 3D engineered constructs from tissue spheroids employing

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54 31 scaffold-free, nozzle-free and label-free magnetic levitational assembly.
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32 Application of magnetic forces in tissue engineering started from series of pioneer studies
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57 33 of Honda and Ito [29]. They coined this approach as a magnetic force-driven tissue engineering.
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59 34 Initially, magnets and associated magnetic fields have been used for the rapid seeding of cells,
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35 labelled by magnetic nanoparticles on the conventional scaffolds. For example, this approach has
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2 1 been used for rapid seeding of labelled endothelial cells on vascular scaffold [30] and for
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4 2 improvement of cell sheets detachment and transfer in cell sheet technology originally developed
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3 by Teruo Okano’s group [31]. The next step involved magnetic forces for 2D patterning of tissue
7 4 spheroids, labelled by magnetic nanoparticles [22-26]. Recently, it was reported that
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9 5 superparamagnetic iron oxide nanoparticles in moderate concentrations are not toxic [32-34] and
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11 6 can even be biodegraded and metabolized into physiological proteins such as ferritins [35].
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7 First, the levitation of large scale non-biological objects was demonstrated [36, 37].
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14 8 MagLev labeled-free approach enabled living material hovering, sorting and fabrication [38]. In
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16 9 these pioneering studies authors approved the use of magnetic levitation to guide the assembly of
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18 10 millimeter to centimeter scale diamagnetic objects in a paramagnetic fluid medium, each
19 11 programmed by shape and density distribution, positioned in the magnetic field gradient,
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21 12 generated by NdFeB magnets. In absence of direct contacts between components, their
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23 13 equilibrium configuration was dependent on the counterbalance of acting magnetic and
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14 gravitational forces. This technique enables positioning of components in 3D.
26 15 The label-free levitational diamagnetic assembly strategy could be used as a powerful
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tool to fabricate soft small living blocks [38]. This strategy allowed for the first time the

30 17 alignment of living blocks in a paramagnetic suspending media for remote 3D assembly. During
31 18 reported magnetic levitation experiments Gd3+ has been used to paramagnetize suspending
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33 19 media. The paramagnetic Gd3+ formulations such as Omniscan have been already approved by
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35 20 FDA for clinical use as a contrast agent for magnetic resonance imaging investigations in
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21 humans. However, the high concentrations of Gd3+ could be potentially toxic for tissue spheroids
38 22 and a certain risk exists for osmotic pressure imbalance due to excessive use of ions in
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40 23 paramagnetic medium. Theoretically, these limitations could be overcome by combination of
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42 24 stronger magnetic fields and smaller density differences between building blocks and medium.
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25 All this enable self-assembly approaches based on magnetic forces that are particularly
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45 26 significant since i) magnetic forces enable contactless manipulation in 3D; ii) the spatial
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47 27 distribution of the magnetic forces can be designed to allow the variation of used arrays of
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49 28 magnets and electromagnets; and iii) a globally applied magnetic field is capable for addressing
50 29 a large number of components in parallel, iv) self-assembly approaches enable scaffold-free
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52 30 biofabrication of 3D tissue constructs [35, 38].

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54 31 Our preliminary experiments confirmed that relatively weak magnetic fields allow
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32 levitational assembly of tissue spheroids only in the presence of toxic Gd3+ concentrations.
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57 33 However, the employment of high gradient magnetic field enables MagLev assembly of tissue
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59 34 spheroids at non-toxic Gd3+ concentrations in paramagnetic medium. Here, we report for the first
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35 time a successful rapid assembly of 3D engineered construct using scaffold-free, label-free and
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2 1 nozzle-free magnetic levitation of tissue spheroids in non-toxic paramagnetic medium. These
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4 2 data provide a proof of concept for new strategies in tissue engineering.
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7 4 Materials and methods
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9 5 Reagents
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11 6 Dulbecco's modified Eagle's medium (DMEM, cat.# 12491-015), fetal bovine serum
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7 (FBS, cat.# 16000-044), antibiotic-antimycotic (cat.# 15240-062), trypsin/EDTA (cat.# 25200-
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14 8 114), phosphate-buffered saline (PBS, cat.# 18912-014) were obtained from Gibco (USA). L-
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16 9 glutamine (cat.# F032) were obtained from Paneco (RF). Glutaraldehyde (cat.# G5882) was
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18 10 obtained from Sigma-Aldrich (USA). CellTiter-Glo 3D kit (cat.# G9682) was purchased from
19 11 Promega (USA). Omniscan (gadodiamide) was purchased from GE Healthcare (Ireland).
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21 12
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23 13 Cell culture
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14 The primary culture of articular sheep chondrocytes was kindly provided by Dr. N.P.
26 15 Omelianenko (N.N. Priorov’s Central Research Institute of Traumatology and Orthopedics,
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Moscow, Russia). Cells were grown in DMEM medium containing 10% FBS, supplemented

30 17 with antibiotic/antimycotic and 2mM L-glutamine. The cells were incubated at 37ºC in a
31 18 humidified atmosphere with 5% CO2 and routinely split at 85-95% confluence. Cell transfer and
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33 19 preparation of single-cell suspensions were performed using mild enzymatic dissociation with a
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35 20 0.25% trypsin/0.53mM EDTA solution. Cells were confirmed free of mycoplasma contamination
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21 according DAPI (Invitrogen, cat. # D1306) staining protocol.
38 22
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40 23 Formation of tissue spheroids using Corning spheroid microplates
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42 24 Tissue spheroids were formed using ultra-low adhesion Corning spheroid microplates
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25 (Corning, cat.# 4520) according to the manufacturer protocol. Briefly, monolayer cells with 95%
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45 26 confluence were rinsed by EDTA solution, harvested from the culture flasks by 0.25%
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47 27 trypsin/0.53mM EDTA and then suspended in cell culture medium. The concentration of the
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49 28 cells was 8x104 per milliliter. 100μl of cell suspensions were dispensed to the wells of Corning
50 29 spheroid microplates. Corning spheroid microplates were incubated at 37ºC in a humidified
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52 30 atmosphere with 5% CO2 for 3 days.

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54 31
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32 Determination of spheroid diameter and roundness distribution
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57 33 Tissue spheroids were biofabricated and captured at 3d day in culture using bright-field
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59 34 imaging at inverted microscope Nikon Eclipse Ti-S, Japan. Spheroid diameters and roundness
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35 were measured using Image J 1.48v software (NIH, Bethesda, MD, USA). Briefly, all original
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2 1 grayscale images were converted to simplified threshold images under the same converting
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4 2 condition and the edges of the spheroids were automatically detected. MinFeret’s diameters of
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3 the detected spheroid edges were measured initially as pixels, and converted to micrometers by
7 4 comparing to a reference length. Roundness was measured using Image J 1.48v shape descriptor
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9 5 and calculated as 4*area/(π*major axis2) [39].
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11 6
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7 Estimation of tissue spheroids viability at different concentrations of gadolinium
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14 8 The viability of tissue spheroids was assessed using the CellTiter-Glo 3D kit according to
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16 9 the manufacturer protocol. Briefly, 3-day-old tissue spheroids with a starting cell number of
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18 10 8000 cells were placed in 0, 50 and 250mM Gd3+ concentrations for 24 hours. Then CellTiter-
19 11 Glo 3D kit was added and luminescence was recorded after 30 min incubation using VICTOR
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21 12 X3 Multilabel Plate Reader (Perkin Elmer, USA).
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23 13
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14 Mechanical testing
26 15 The mechanical properties of tissue spheroids were measured by a micro-scale parallel-
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plate compression testing system Microsquisher (CellScale, Canada) with associated

30 17 SquisherJoy software. Tissue spheroids with a starting cell number of 8000 cells were formed
31 18 using Corning spheroid microplates. They were cultured 3 days and then placed in 0, 50 and
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33 19 250mM Gd3+ concentrations for 24 hours prior to mechanical characterization. For mechanical
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35 20 testing spheroids were placed in a PBS-filled bath at 37 ºC and compressed to 50% deformation
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21 in 20 sec. The microbeams with diameters 152.4μm (recommended max force 57 mN) and 304.8
38 22 μm (recommended max force 917 mN) were employed depending on the stiffness and sensitivity
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40 23 required to measure tissue spheroids from different cell types. The force-displacement data
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42 24 obtained from the compression test were converted to stress-strain curves and the lower portion
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25 of the curve (0-20% strain) was used to obtain a linear regression line and estimate the Young’s
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45 26 modulus. In each group eight samples of spheroids were measured.
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47 27
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49 28 Spheroid fusion assay
50 29 Spheroid fusion assay was performed using Corning spheroid microplates (Corning, cat.#
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52 30 4520). Pairs of 3-day-old tissue spheroids with a starting cell number of 8000 cells were placed
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54 31 in contact in 0, 50 and 250mM Gd3+ concentrations and incubated for 7 days. Bright-field images
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32 of spheroid doublets were obtained at points 0 h, 4 h, 6 h, 1 day, 2 days, 3 days, 4 days and 7
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57 33 days using Nikon Eclipse Ti-S microscope. Contact length, intersphere angle and doublet length
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59 34 were measured using Image J 1.48v software (NIH, Bethesda, MD, USA) and plotted as a
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35 function of time using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA).
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2 1 Doublet length (Fig. 5) was normalized to initial doublet length. Measurements for each
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4 2 parameter were reported as mean ± S.E.M.
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7 4 Electrospinning of polyurethane
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9 5 Polyurethane was kindly provided by Dr. Xuejun Wen (EG-85A, Lubrizol, USA).
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11 6 Electrospinning of microfibrous polyurethane matrix was performed using commercial
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7 Professional Electrospinning Lab Device (Yflow, Spain). Polyurethane was dissolved to 17%
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14 8 concentration in solvents containing 40% N,N-dimethylformamide and 60% tetrahydrofuran.
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16 9
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18 10 Scanning Electron Microscopy (SEM)
19 11 Tissue spheroids and constructs made from chondrospheres were placed on the surface of
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21 12 electrospun polyurethane matrix and allowed to attach during 6 hours. Subsequently samples
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23 13 were fixed with 2.5% glutaraldehyde/PBS, dehydrated through ethanol series and then were
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14 dried in a critical point dryer (HCP-2, Hitachi Koki Co. Ltd., Japan). The samples were
26 15 transferred on a stub of metal with adhesive surface, coated with gold using ion coater (IB-3,
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EIKO, Japan) and then observed using the microscope JSM -6510 LV (JEOL, Japan).

30 17
31 18 Tissue spheroids morphology
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33 19 Tissue spheroids were fixed with 2.5% glutaraldehyde/PBS and then treated with 1%
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35 20 osmium tetraoxide/PBS. The samples were dehydrated by soaking in a series of solutions of
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21 increasing concentrations of ethanol, stained with 1% uranyl acetate/70% ethanol and finally
38 22 embedded to araldite-epon mixture. Semi-thin sections were prepared on a LKB-III ultratome
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40 23 (Sweden), stained with 1% toluidine blue and then examined using a light microscope (Leica
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42 24 DM 2500, Germany) equipped with a digital camera (Leica DFC 290, Germany).
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45 26 Tissue construct histology
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47 27 The constructs, assembled from chondrospheres in magnetic field, were fixed in 10%
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49 28 buffered formalin (pH 7.4) for 24 hours and then embedded in paraffin with a melting point of
50 29 +54°C (Biovitrum, Russia). Serial sections with a thickness of 5μm were made with microtome
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52 30 Microm HMS 740 (ThermoFisher Scientific, USA), routinely stained with hematoxylin and
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54 31 eosin (Biovitrum, Russia) and then covered with Bio-Mount medium (Bio Optica Milano S.P.A.,
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32 Italy) before histological examination.
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59 34 Estimation of Spheroid Cell Density
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2 1 Spheroid cell density was estimated by analysis of semi-thin sections of tissue spheroids
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4 2 using Image J 1.48v software (NIH, Bethesda, MD, USA).
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7 4 Determination of polystyrene bead density
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9 5 The density of polystyrene beads (Polyscience, Inc. USA, cat.# 64235) was determine by
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11 6 means of equating the force of gravity with buoyancy force. We put polystyrene beads with
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7 diameter 170μm in the container with deionized water, and then the dextrose powder was added
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14 8 to the container followed by the thorough mixing. Gradual adjusting of the dextrose
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16 9 concentration in the solution provided floating of the beads in the water volume without sinking
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18 10 or emerging at the surface. Thus, we obtained the polystyrene beads density equal to 1.0405
19 11 g/cm3 .
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23 13 Surface evolver simulation
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14 The fusion behaviour of tissue spheroids was illustrated using the open source software
26 15 Surface Evolver, developed and described in details earlier [40]. Tissue spheroids were
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approximated and modelled as ball-like liquid droplets of standardized size and volume. The

30 17 initial positions of closely contacting tissue spheres were random, then evolved by gradient
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18 descent to minimize distance between centers subject to centers being at least a diameter apart.
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33 19 The progressive process of fusion of tissue spheres was modeled by iterating gradient descent to
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35 20 minimize the surface energy subject to the constraint of constant spheroid volumes, until
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37 21 movement stopped. The evolution script created interfaces between spheroids where spheroids
38 22 touch. The configuration reached is a local minimum of energy, but not necessarily a global
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40 23 minimum. The progressive changes in the shape of single spheroids contacted with each other
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42 24 inside forming compacted tissue constructs have been visualized. The simulations have been
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25 performed for 40 tissue spheroids.
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47 27 Magnetic experimental setup
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49 28 To perform the experiment we designed custom laboratory installation with construction
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29 shown on Fig.1 (a, d). The installation consists of 2 CMOS digital cameras (DMK41AU02, The
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52 30 Imaging Source Europe GmbH, Germany), light sources, lens system (Optem ZOOM 70XL,
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54 31 Qioptiq, Germany), which are mounted on 3-axis positioning system assembled from 3 linear
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56 32 stages (TSX-1D, Newport Corporation, USA), magnet holding system and 2 ring-shape NdFeB
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57 33 magnets with cutouts for video capture (Fig. S1). The external diameter of magnets is 85 mm;
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59 34 the internal diameter is 20 mm; thickness (height) is 24 mm. Magnets are assembled in such a
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35 way that they oriented to each other with the same poles. This experimental setup was placed in
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2 1 the incubator to ensure the appropriate temperature regime (37°C) during the process of the
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4 2 constructs assembly.
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3 Non-homogeneous magnetic field with orifice in the center of the magnets was created in
7 4 the axial hole of the magnetic installation (working zone). The distribution of the magnetic
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9 5 induction values in the working area vertical section is shown in the 3D model graph (Fig. 1c). A
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11 6 transparent glass cuvette with dimensions of 12 x 12 x 50 mm we inserted into the hole of the
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7 magnet system (Fig. S1). The cuvettes were filled with paramagnetic liquid contained
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14 8 diamagnetic particles: polystyrene beads or tissue spheroids. The paramagnetic liquid consisted
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16 9 of the DMEM medium and range of Gd3+ concentration: 0, 50, and 250mM. The process of
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18 10 levitational assembly for polystyrene beads and tissue spheroids was recorded by two video
19 11 cameras with 1x and 3x optical magnification. After tissue spheroids assembling, resulted
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21 12 constructs continued to levitate in a magnetic field for 24 hours to full complete the fusion
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23 13 process.
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14 The principle of operation of experimental installation implicates the creation of a local
26 15 microgravity zone where the effects of all acting on diamagnetic objects forces are compensated.
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In our setup, the directed upward Archimedes force, the directed downward force of gravity, and

30 17 the directed toward the local minimum of the magnetic field magnetic force simultaneously act
31 18 on the object under static conditions. The magnetic forces moment appears only if the magnetic
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33 19 field is non-homogeneous. Then the effective magnetic force [41], acting on the object in non-
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35 20 homogeneous magnetic field will be equal to the volume integral:
36 𝜒𝜒𝜒𝜒
37 21 F= ∇(𝑩𝑩2 ),
2
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39 22 where for paramagnets 𝜒𝜒 > 0, while for diamagnets 𝜒𝜒 < 0. The sign determines the
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23 direction of the magnetic force action.
42 24 As a result, diamagnetic objects will be pushed out into an area with lower field strength
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44 25 (magnetic trap) under the action of a magnetic force. In Earth’s gravity condition the
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46 26 equilibration of objects occurs at a certain distance from the local minimum of the magnetic
47 27 field.
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51 29 Computer simulation of magnetic field
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53 30 The simulation of three-dimensional non-homogeneous static magnetic field in a
54 31 paramagnetic medium from two permanent magnets we performed using "ANSYS
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56 32 Electromagnetics Suite" software for Maxwell3D. The characteristics of magnetic field used in
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58 33 the simulation were as follows: relative permeability paramagnetic medium 1.00004135; and
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34 material grade of NdFeB magnet N38 (VG=1.21 TL).
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4 2 Molecular dynamics (MD) simulation of dynamics of the polystyrene particles in the
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3 cusp magnetic trap
7 4 The MD modeling was carried out as described previously [42-44]. We assumed that all
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9 5 particles in MD calculations are spherical, having the same size and mass mp. For modeling
10
11 6 dynamics of the diamagnetic polystyrene particles in the cusp magnetic trap, we have
12
7 numerically solved the system of the Newton equations for all the particles (1 < k < N)
13

cri
14 d 2rk r
15 8 mp 2
= ∑ F ( rkl ) kl + FkB + f k + f g + fb , (1)
16 dt l rkl
17
18 9 where rk is the position of the center of a particle k, rkl= rk − rl . The first term in the right-hand
19
20 10 part of Eq. (1) is the Lennard-Jones interaction with other particles, the second term is the

us
21
22 11 interaction with the magnetic field of the trap (Fig. 1c ), the third term is the force of viscous
23
12 friction against the continuum liquid which is determined by the Stokes formula f k = −3πdηu k , η
24
25
26
13 is the viscosity of Gd3+ suspension, d is the particle diameter and uk = drk / dt is the particle
an
27
28
14 velocity relative to the stationary liquid, the fourth and the fifth terms is a force of gravity f g
29
30 15 and buoyancy force f b acting on the particle. Numerical simulation was performed for N = 100.
31
32 16 The simulation was performed for polystyrene beads and tissue spheroids with densities
dM
33 17 ρ = 1.0405 g/cm3 and ρ = 1.05 g/cm3, respectively. At the first step, particles were distributed
34
35 18 randomly within the computational domain with initial zero velocity. The computational domain
36
37 19 corresponds to the internal volume of the experimental cell - 12×12×47 mm.
38
39 20
40
21 Data analysis
41
42 22 Statistical data was analyzed using GraphPad Prism software (GraphPad Software, Inc.,
43
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44 23 La Jolla, CA) and represented as mean ± S.E.M. The Analysis of Variance (ANOVA) test was
45
46 24 used to find the significant differences between the means of the three groups with P< 0.0001.
47 25
48
49 26 Results
50
51 27 Magnetic experimental setup
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52
53
28 We created a magnetic installation (Fig. 1d), which generates a magnetic field in the
54 29 working volume with a very high (calculated value is up to 2.2 T/cm) gradient along the vertical
55
56 30 axis. The distribution of magnetic induction, created by the system of magnets, is shown in Fig.
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57
58 31 1 (b, c, e). We used molecular dynamics modeling to estimate the time, shape and height of
59
32 constructs assembly in a magnetic field.
60

10
Page 11 of 28 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1

1
2 1 The fusion dynamics modeling resulted in the graph (Fig. 1f), which shows the relation
3
4 2 between the projection on the vertical axis of the resultant force, acting on particles, tissue
5
6
3 spheroids or polystyrene beads, during the experiment, and the vertical coordinate in the
7 4 laboratory installation system of axes.
8

pt
9 5 We used polystyrene beads to test the operations and debugs for all modes. They proved to
10
11 6 be sufficiently good physical analogues of tissue spheroids for such parameters as magnetic
12
7 susceptibility and density. The use of tissue spheroids for routine testing of assembly modes was
13

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14 8 time and cost consuming, so we used polystyrene beads as their substitutes. Indeed, beads and
15
16 9 tissue spheroids have the similar parameters of density and magnetic susceptibility. After initial
17
18 10 determination of the optimal magnetic field configuration, we continued our experiments with
19 11 tissue spheroids.
20

us
21 12 The construct’s center of mass corresponds to the point, where the resultant force turns into
22
23 13 zero, thus forming levitation point. According to the graph (Fig. 1f) this point has the following
24
25
14 coordinates in the vertical axis: 3.2 mm for polystyrene beads and 5.6 mm for tissue spheroids.
26 15 The vertical coordinate of construct’s center of mass was calculated by means of video analysis.
27
28
29
16
an
The coordinates, defined in such way were as follows: 3.9 mm for polystyrene beads, 5.1 mm for

30 17 tissue spheroids (Fig. 6). Results of our simulation are in a good agreement with experiments.
31 18
32
dM
33 19 Biofabrication of tissue spheroids
34
35 20 Tissue spheroids were formed from primary sheep chondrocytes using ultra-low adhesion
36
37
21 Corning spheroid microplates (Fig. 2a). Their morphology was estimated using bright-field
38 22 microscopy and SEM. We’ve used 3-day-old spheroids for construct biofabrication. Tissue
39
40 23 spheroids had well-defined borders and spherical shape at 3d day in culture (Fig. 2b). In order to
41
42 24 investigate morphology and surface characteristics of tissue spheroids we analyzed them by
43
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25 SEM (Fig. 2c). The surface of spheroids was formed by closely packed cells. Standard size and
44
45 26 shape of tissue spheroids is an essential prerequisite for their use in bioprinting as building
46
47 27 blocks. Spheroid diameters (Fig. 2d) and roundness (Fig. 2e) were measured after 3 days of
48
49 28 culture. The average spheroid diameter was 346±16.58 µm. The average spheroid roundness was
50 29 0.907±0.047. The standard deviations were <10% of the mean value for both diameter and
51
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52 30 roundness. These results revealed that tissue spheroids had uniform standardized geometry
53
54 31 and could be used for biofabrication of three-dimensional tissues.
55
56
32
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57 33 Histological analysis and viability of tissue spheroid

58
59 34 A histological analysis on semi-thin sections of 3-day-old tissue spheroids was performed
60
35 in order to estimate possible toxic effects and to determine an optimal nontoxic Gd3+
11
 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1 Page 12 of 28

1
2 1 concentration (Fig. 3a). In non-treated conditions, chondrospheres demonstrated high cell
3
4 2 density, swirl-like cell arrangement and intensive staining of cytoplasm. In 50mM Gd3+ solution
5
6
3 cell density did not change, but cytoplasm staining was slightly reduced. Finally, at toxic 250mM
7 4 Gd3+ concentration, cell density dramatically reduced, that led to manifestations of cell death and
8

pt
9 5 apoptosis in form of pycnotic nuclei and accumulation of extracellular debris.
10
11 6 The viability of cells within tissue spheroids was analyzed by measuring the luminescent
12
7 signal generated by luciferin-luciferase interconnection as a function of cytoplasmic ATP
13

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14 8 concentration. Fig. 3b shows that tissue spheroids in 50mM Gd3+ solution remained viable after
15
16 9 24 hours of incubation, while 250mM Gd3+ concentration revealed significant toxic effect. High
17
18 10 statistically significant difference, p<0.0001, in cell density corresponded to 85% in 50mM Gd3+
19 11 solution compared to 100% in 0mM Gd3+ solution, while the cell density at 250mM Gd3+
20

us
21 12 concentration was 40% (Fig. 3c). Thus, cell counting showed that the increase of Gd3+
22
23 13 concentration leads to the reduction in the cell density.
24
25
14
26 15 The Mechanical Properties of Tissue Spheroids
27
28
29
16
an
The influence of gadolinium Gd3+ on mechanical properties of tissue spheroids was

30 17 estimated by tensiometry using parallel plates modification. As shown in Figure 4, the

31 18 mechanical properties of spheroids strongly depended on concentration of gadolinium Gd3+. The
32
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33 19 values of spheroids elastic modulus in 0 and 50mM Gd3+ solutions were 2.91±0.15 kPa and
34
35 20 2.95±0.1 kPa, correspondingly. The increase of Gd3+ concentration to 250mM resulted in
36
37
21 decrease of elastic modulus value to 0.14±0.05 kPa. This observation can be explained by low
38 22 viability of tissue spheroids in 250mM Gd3+ solution.
39
40 23 Fig. 4a shows the stress-strain diagram obtained from the compression load-displacement
41
42 24 curve. The sharp rise of the curve in the area of high strain values is associated with an increase
43
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25 of the tissue spheroid cross section. Fig. 4c shows the stages of chondrosphere compression
44
45 26 process between two parallel plates. When compressing up to 20% of the original diameter, an
46
47 27 increase of the cross section does not occur. The stress-strain curve changes virtually linearly
48
49 28 before this strain; in this section, we calculated the Young's modulus. According to our results,
50 29 the nontoxic concentration 50mM Gd3+ in the growth medium does not affect the tissue
51
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52 30 spheroids Young's modulus at all, which apparently means no significant changes in the internal
53
54 31 structure of the tissue spheroid (Fig. 4b). Finally, at a toxic concentration 250mM Gd3+, the
55
56
32 Young's modulus sharply decreases by ~ 93%, indicating that at a given concentration of
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57 33 gadolinium salts irreversible changes occurred within the cells, that clearly affects the
58
59 34 mechanical properties of the tissue spheroid.
60
35
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1
2 1 The Fusion of Tissue Spheroids
3
4 2 To investigate tissue spheroids fusion dynamics at different Gd3+concentrations,
5
6
3 spheroids in pairs were placed in contact with each other in the same well of low adhesion
7 4 microplate and allowed to fuse for 7 days. The kinetics of tissue spheroid fusion was monitored
8

pt
9 5 and analyzed daily in bright-field under inverted microscope. Contact length, intersphere angle
10
11 6 and doublet length were measured according to the method described by Susienka et al. [45]
12
7 (Fig. 5a). As shown in Figure 5b, for spheroid pairs in 0 and 50mM Gd3+ solutions contact
13

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14 8 lengths and intersphere angles increased as a function of time and doublet lengths shortened
15
16 9 gradually from first day in culture. Spheroid doublet in 0mM Gd3+ solution shortened faster than
17
18 10 spheroid doublet in 50mM Gd3+ solution. At the seventh day of incubation the intersphere angle
19 11 increased up to 179º, indicating complete spheroid fusion. Spheroid pair in 250mM Gd3+
20

us
21 12 solution continued to fuse during first 24 hours and then no further progressive aggregation was
22
23 13 observed. This observation confirmed also the high cytotoxicity of 250mM Gd3+ concentration.
24
25
14 To check the possibility of microtissue construct biofabrication at different
26 15 concentrations of Gd3+, we placed 30 chondrospheres in the same well of non-adhesive
27
28
29
16
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microplate and allowed fusion for 48 hours (Fig. 5c). At 0 and 50mM Gd3+ concentrations the

30 17 formation of compact constructs was observed after 2 days of incubation, while no construct was
31 18 formed in 250mM Gd3+ solution.
32
dM
33 19
34
35 20 Assembly of constructs in a magnetic field
36
37
21 We performed construct assembly in magnetic field using two types of building blocks.
38 22 The time length for tissue spheroids assembly was shorter by 20% ± 1.5% compare to one for
39
40 23 polystyrene beads assembly due to the difference in the magnetic susceptibility of diamagnetic
41
42 24 materials. Meanwhile, the height of the assembled constructs was lower by 1.2mm ± 0.2 mm
43
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25 because the spheroids density is higher than the polystyrene beads density, and, as a result, the
44
45 26 gravity force acts more strongly on them. It means that the force compensation will occur in the
46
47 27 area where the magnetic force is higher, that is closer to the magnet surface. The density of the
48
49 28 chamber medium was assumed ρ = 1.015 g/cm3. Considering the near-spherical configuration of
50
51 29 the magnetic field in the trap we can conclude that the final form of the cluster obtained in MD
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52 30 simulation should be an oblate body with relation between the semi axes a y / ar = 1 .
53
54
31 In MD simulations for the formation of cluster from polystyrene particles with diameter d
55
56 32 = 170µm and tissue spheroids of diameter d = 350µm, we set χ = −0.65⋅10−6 cm3/g and
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57
58 33 −0.72⋅10−6 cm3/g, respectively. For sufficiently large N the shape of the cluster became almost
59
60 34 independent of N, and the ratio of the thickness and diameter of the cluster H c / Dc was close to

13
 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1 Page 14 of 28

1
2 1 the theoretical value of the relation between the semi axes a y / ar = 1 . Experimental values of the
3
4 2 ratio H c / Dc for polystyrene and tissue spheroids were almost equal and corresponded to the
5
6 3 theoretical values.
7
8 4 Configurations of the polystyrene beads and tissue spheroids clusters obtained

pt
9
10 5 experimentally and in MD simulation are shown in Fig. 6a and presented in video S1 and S2.
11 6 The shape and size of the clusters were similar. Inside the formation area, the force of gravity,
12
13 7 acted on the particles, was counterbalanced by the magnetic force of the trap and the buoyancy

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14
15 8 force. The center of the mass for the polystyrene particles structure formed by MD simulation
16
17
9 located by a distance 3.2mm downward from the center of the trap. For tissue spheroids, this
18 10 displacement was up to 5.6mm.
19
20 11 SEM analysis proved the fusion of tissue spheroids into the compact 3D tissue construct

us
21
22 12 (Fig. 6e). Despite the presence of some grooves and pits, corresponding to separate
23
13 chondrospheres, the ongoing tissue fusion process was obvious, revealed by gradual smoothing
24
25 14 of the construct surface Mathematical modeling and computer simulation using Surface Evolver
26
27
28
29
15
16
an
software demonstrated that the formation of grooves is unavoidable in case of random packing
(Fig. 7a,b,c). Thus, the formation of holes and empty space could be explained by either the
30 17 incomplete fusion process or by a chondrospheres rigidity. Our previous experiments involved
31
32 18 softer tissue spheroids, namely, embryonic mouse thyroid gland explants, these showed complete
dM
33
34 19 fusion in rounded and compact tissue construct without any holes and empty space [13].
35
36
20 We performed histological analysis to visualize chondrospheres status and fusion in
37 21 biofabricated tissue construct (Fig. 7d). It was demonstrated, that viable chondrocytes were
38
39 22 tightly packed inside tissue spheroids. The spheroids fusion kinetics within biofabricated 3D
40
41 23 tissue construct was different. As results, there were some areas of complete fusion and there
42 24 were some gaps or incomplete fusion in tissue which probably reflects random initial packing of
43
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44 25 chondrospheres. Similar void areas have been shown previously during cell aggregates fusion on
45
46 26 pre-stretched electrospun synthetic matrices [20] and also during fusion of chondrospheres
47
48
27 biofabricated from cells labeled with iron oxide magnetic nanoparticles [46, 47].
49 28
50
51
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29 Discussion
52
53 30 The main outcome of our study is a proof of concept for magnetic levitational assembly
54
31 of microtissue constructs applying viable tissue spheroids.
55
56 32 The horizons of magnetic levitational assembly have been demonstrated in several
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57
58 33 pioneering publications [36-38]. In early studies, authors used predominantly non-biological
59
60 34 microobjects, such as plastic beads, cells on the beads and cells within hydrogel blocks. We

14
Page 15 of 28 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1

1
2 1 reported for the first time the successful rapid biofabrication of millimeter scale microtissue
3
4 2 constructs from chondrospheres applying magnetic levitational assembly.
5
6
3 The Earth’s gravity is one of the most serious hindrances for MagLev assembly of
7 4 biological constructs. One way to overcome it is to use strong magnets with addition of
8

pt
9 5 paramagnetic substances, i.e. Gd3+ into working solution. However, the toxicity of paramagnet
10
11 6 concentration enabling the levitation of objects comprises the feasible limitation of approach. In
12
7 our study, we designed an optimal form of permanent magnets and estimated their mutual
13

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14 8 emplacement using the MD simulation that allowed us to perform levitational assembly with
15
16 9 nontoxic concentration of the paramagnet in the culture medium. The rate of construct assembly
17
18 10 and its geometrical shape were pre-calculated. Thus, a rather simple, but original magnetic
19 11 system was created, which provided generation of a magnetic field with a gradient of up to 2.2
20

us
21 12 T/cm with a local minimum at the center of the working area. In the course of an experiment, we
22
23 13 obtained the data on the construct assembly kinetics and its spatial characteristics, which
24
25
14 confirmed the validity of the calculated magnetic field distribution in the magnetic system
26 15 working area.
27
28
29
16
an
Our data demonstrated that the use of permanent magnets with defined shape and relative

30 17 position significantly accelerates the assembly of both polystyrene beads and tissue spheroids.
31 18 Quantitative estimation of cytoplasmic ATP production to prove cell’s viability, histological
32
dM
33 19 analysis and confirmed tissue spheroids capacity for fusion have justified the vitality of
34
35 20 chondrospheres, exposed to 50mM of Gd3+ in course of magnetic levitation.
36
37
21 For biofabrication of tissue-engineered constructs of much larger sizes it is necessary
38 22 either to use superconducting magnets, providing the required magnetic field gradient throughout
39
40 23 the working volume, or apply a higher concentration of the paramagnetic solution. However, per
41
42 24 our data, the high concentration of the paramagnetic induces the undesirable cytotoxic effects.
43
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25 Achievement of sufficient magnetic field gradient in a larger working volume is still unsolved
44
45 26 and challenging bioengineering task.
46
47 27 Alternative approach involves the use of space microgravity which will allow
48
49 28 significantly reduce the concentration of Gd3+. Our preliminary calculations, mathematical
50 29 modeling and computer simulations strongly suggest that application of strong magnetic field in
51
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52 30 space microgravity conditions will allow to reduce significantly the concentration of Gd3+ and to
53
54 31 increase the size of biofabricated tissue constructs. After rapid levitational assembly of spheroids
55
56
32 in space, Gd3+ could be simply washed out and replaced by conventional cell culture media for
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57 33 post-processing, thus providing biofabricated organoids suitable for prolonged space missions.
58
59 34 In our study, we have chosen to use chondrospheres, fabricated from articular
60
35 chondrocytes. Previously it has been shown that closely placed chondrospheres both loaded and
15
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1
2 1 unloaded with magnetic nanoparticles can fuse into large size cartilage constructs using scaffold-
3
4 2 free approach [26, 48]. Moreover, the treatment of cartilage defects by chondrospheres, capable
5
6
3 to attachment and spreading on injured surface of cartilage had already been demonstrated in
7 4 several independent preclinical and clinical studies [49-51]. However, despite the many positive
8

pt
9 5 examples for medical application of magnetic, especially, iron oxide based, nanoparticles, their
10
11 6 possible cytotoxicity in vivo is still a challenge [52, 53]. Our data demonstrates that magnetic
12
7 levitation provides simple and rapid way for scaffold-free and label-free tissue construct
13

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14 8 bioassembly. Recently, the systematical investigation of the solid scaffold-free and label-free
15
16 9 acoustic levitation has been initiated by several groups as an additional potential alternative
17
18 10 technology for conventional solid scaffold-based or label-based 3D patterning [54, 55]. In this
19 11 context, one could assume that combination of magnetic and acoustic levitation will potentially
20

us
21 12 enable the rapid assembly of tissue and organ constructs with increased complexity. Specially
22
23 13 designed system of repulsive acoustic holes with multiple spatially arranged acoustic field
24
25
14 generators can ensure successful implementation of this intriguing approach. Similarly, the
26 15 employment of tissue spheroids with more advanced intrinsic properties and compositions such
27
28
29
16
an
as heterocellular aggregates, lumenized vascular tissue spheroids, pre-vascularised organo-

30 17 specific aggregates and sacrificial vascular tissue spheroids will ensure highly desirable
31 18 vascularization state and dramatically enhance the level of complexity of biofabricated tissue and
32
dM
33 19 organ constructs [56-59].
34
35 20 Theoretically, scaffold-free approach implies the absence of any temporal support such as
36
37
21 biodegradable biomaterials or scaffolds. Taking this, it looks like that we indeed employed
38 22 scaffold-free or at least biomaterials-free approach. However, magnetic field as well as acoustic
39
40 23 or electric fields provides temporal and removable support and could be considered as a new
41
42 24 form of scaffold. In order to escape possible confusion, we propose to call this new type of
43
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25 temporal support-based employment of physical fields as a “scaffield”.

44
45 26 Thus, presented scaffold-free, label-free and nozzle-free magnetic levitational assembly
46
47 27 in possible combination with acoustic hoist of tissue spheroids and other forms of physical force-
48
49 28 based 3D patterning of cell aggregates can potentially be a novel and more efficient approach for
50 29 rapid manufacturing of complex tissue and organ constructs. Design, development and
51
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52 30 implementation of new types of tissue and organ bioassemblers, based on the proposed concept
53
54 31 of “scaffield” could dramatically advance the emerging field of organ and tissue biofabrication.
55
56
32
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57 33 Conclusion
58
59 34 Scaffold-free, label-free and nozzle-free biofabrication technology applying magnetic
60
35 levitational assembly has been demonstrated. 3D living tissue constructs have been biofabricated
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Page 17 of 28 AUTHOR SUBMITTED MANUSCRIPT - BF-101339.R1

1
2 1 from chondrospheres in paramagnetic medium using non-toxic Gd3+concentration. The
3
4 2 introduced technology can became a powerful biofabrication tool that enables rapid assembly of
5
6
3 various tissues and organs.
7 4
8

pt
9 5 Acknowledgements
10
11 6 Authors thank Prof. Xuejun Wen (Virginia Commonwealth University, USA) for
12
7 providing polyurethane for electrospinning.
13

cri
14 8
15
16 9 References:
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18 10 1. Lin RZ, Chang HY. Recent advances in three-dimensional multicellular spheroid culture
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21 12 2. Achilli T-M, Meyer J, Morgan JR. Advances in the formation, use and understanding of
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38 5 construct assembly from polystyrene beads and chondroshperes. (e) Scanning electron
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