Optimization of Femtosecond Laser Polymerized Structural Niches to Control Mesenchymal Stromal Cell Fate in Culture

: We applied two-photon polymerization to fabricate 3D synthetic niches arranged in complex patterns to study the effect of mechano-topological parameters on morphology, renewal and differentiation of


Introduction
Recent advances in stem cell biology have opened up new therapeutic strategies for a variety of incurable diseases, but the ultimate clinical success of such therapies lies entirely on our ability to efficiently control and manipulate stem cell fate and produce therapeutic cells in large, pharmaceutically relevant scales.Recent developments towards this goal consist in the fabrication of culture substrates to be used as "synthetic niches" for engineering stem cells [1].These are synthetic polymeric systems mimicking individual aspects of the stem cell interaction with the extra-cellular microenvironment, for example material properties (chemical functionality, tissue-mimetic modifications such as mineralization, etc.), and spatiotemporal variations of the presence of specific bioactive agents (growth and differentiation factors, nutrients, etc.).Synthetic niches are useful both for understanding stem cell behavior under three-dimensional (3D) biomimetic conditions, and to develop new strategies for long term maintenance of pluripotency or to promote lineage-specific differentiation into therapeutic cells.For example, hydrogel-based synthetic niches were recently used to study the synergistic role of matrix microenvironment (type, architecture, composition, stiffness) and signaling molecules (type, dosage) to direct murine embryonic stem cell (ESC) differentiation into specific neural and glial lineages [2].
Within this context, a potential strategy for guiding stem cell behavior is the employment of purely mechanical cues [3].For example, spontaneous lineage commitment has been induced in mesenchymal stromal cells (MSCs), by culturing them on collagen-coated polystyrene microbeads [4], or on hydroxyapatite/chitosan nanofibrous scaffolds [5].Altering cell culture conditions from 2D to 3D results in alteration of cell adhesion, causing major remodeling of the cellular cytoskeleton.This, in turn, induces alterations in nuclear shape, mediated by the traction transmitted by the filamentous actin cytoskeleton [6].Nuclear shape is emerging as a primary factor orchestrating complex stem cell behaviors, including maintenance of the cell multipotency and lineage commitment [7].Thus, a precisely controlled mechano-topological environment is becoming increasingly recognized as a crucial factor in stem cell culture, because it allows exploring the relationship between the 3D structural interaction of the adhered cell with its physical microenvironment, and its response.
Direct laser writing (DLW) by two photon polymerization (2PP) [8][9][10][11] exploits focused femtosecond laser pulses to induce photopolymerization within the focal volume, following a nonlinear two-photon absorption mechanism.2PPis the only fabrication technique in which the scaffold geometry may be controlled at the cell scale (10 μm) and with a very high spatial resolution (less than 1 μm).Non-biodegradable "structural" materials used to fabricate scaffolds by 2PPinclude vinyls [12], epoxies [13], acrylates [14] and hybrid inorganic-organic materials [15], many of which have proven to be biocompatible in the cell culture environment [16].In recent work, we have developed structural microscaffolds, or niches, of various complex 3D architectures, which were laser-written directly on the glass bottom of the cell culture chamber, using a photoresist consisting of a solgel-synthesized silicon-zirconium hybrid inorganic-organic [17].In this way, we were able to select the niche microarchitecture most favoring spontaneous homing and proliferation of MSCs, among the various ones tested.However, deeper insight into the behavior of cells proliferating inside the niches was hindered by a strong fluorescence background, generatedby the polymeric structure itself that masked the signals from biological markers.
Here, we report on morphology, proliferation and differentiation of MSCs in long term culture inside "structurally" biomimetic synthetic niches.A photosensitive resin with low autofluorescence enabled immunofluorescence diagnostics inside the microscaffolds.A different behavior of the cells is observed depending on the environment where they proliferate: a lineage commitment is observed in 2D and 2.5D regions while a preservation of their multipotency is noted in 3D engineered scaffolds.This striking effect is attributed to a reduced deformation of the cell nuclei following an isotropic cytoskeletal tension state, and could find application in the development of novel customized cell therapies.

Two-Photon Polymerization of 3D Niches
Scaffolds were directly two-photon polymerized in the SZ2080 photoresist (Maria Farsari, IESL-FORTH, Heraklion, Greece) with 1% concentration of Irg photoinitiator (Irgacure 369, 2-Benzyl-2-dimethylamino-1-(4-morpholinophenyl)-butanone-1).The laser used for 2PP was a regeneratively amplified Yb:KGW system (Pharos, Light Conversion, Vilnius, Lithuania) with 200-fs pulse duration, 515-nm wavelength (frequency doubled), focused with a 1.4-NA oil immersion lens (Plan-APOCHROMAT, 100× oil immersion, Carl Zeiss, Oberkochen, Germany).The collimated beam diameter D before the lens was 5 mm, slightly overfilling the 4.5-mm clear aperture (CA), allowing us to exploit the full NA of the objective.The transmission of the lens was measured to be 50%, which is expected given the 69% transmission quoted by Zeiss and the ratio of the area of the aperture to that of the overfilling beam (CA/D) 2 = 0.8.The theoretical spot size 2w 0 was 0.3 μm for our laser having a beam quality of M 2 = 1.3.
Optimum fabrication conditions were 1-mm/s writing speed, 0.75-mW average power (before the objective) and 200-kHz repetition rate.After the focusing objective, this corresponds to a pulse energy of 1.9nJ, a peak fluence of 5.4 J/cm 2 and a peak intensity of 27 TW/cm 2 [18].Computer-controlled, 3-axis motion stages (ABL-1000, Aerotech, Pittsburgh, PA, USA) interfaced by CAD-based software (ScaBase, Altechna, Vilnius, Lithuania) with an integrated acousto-optic modulator (AOM) were used to translate the sample relative to the laser to form the desired 3D microarchitectures.The microscaffolds were laser written directly onto circular glass cover slips of 150 μm thickness and 12 mm diameter (BioOptika, Milano, Italy).
Microscaffolds were 30 μm high and 90 μm × 90 μm in transverse dimensions and consisted of a lattice of interconnected lines, with a graded spacing between 10 and 30 μm transversely and a uniform spacing of 15 μm vertically.The 3D scaffold was surrounded by four outer confinement walls formed by horizontal lines spaced by 7.5 μm, resulting in gaps of 2 μm.

Cell Isolation
Unless otherwise specified, all chemicals and chemofluorescent markers were purchased from Sigma-Aldrich; immunofluorescent markers from Thermo Scientific; cell culture media and plastics from Euroclone.All protocols comply with institutional ethical use protocols for laboratory animals.
We used primary rat MSCs to study self-renewal and differentiation within the 2PP-engineered microscaffolds.Bone marrow was obtained from 2-month-old Sprague-Dawley (CD) rats.Briefly, rats were sacrificed and femurs and tibias were aseptically removed.Bone marrow was flushed from the shaft of the bones with α-Minimum Essential Medium (Invitrogen-Gibco) containing 5% fetal calf serumplus 100 U/mL penicillin G and 0,1 mg/mL streptomycinand then filtered through a 100-μm sterile filter to produce a single-cell suspension.MSCs were recovered from bone marrow by their tendency to adhere tightly to plastic culture dishes.Filtered bone marrow cells were plated in α-MEM supplemented with 20% FCS and 1% penicillin/streptomycin and allowed to adhere for 24 h.Non-adherent cells were then removed.Medium was changed regularly every 3 days until confluence.Adherent cells were detached by trypsin-EDTA (0.5-0.2 g/L; Invitrogen, Carlsbad, CA, USA), counted and cryo-preserved in α-MEM supplemented with 30% FCS and 5% dimethyl sulfoxide (DMSO) until use.After resuscitation, cells were plated and cultured until semi-confluence in standard flasks in complete medium.

Characterization of the Freshly Isolated MSCs
The isolated cells were characterized as described in detail in [19].Briefly, fluorescence-activated cell sorting (FACS) analysis revealed that MSCs were negative (98% negative cells) for the hematopoietic marker CD45 (anti-rat CD45 Ab, BD Pharmingen, San Jose, CA, USA).Cells were also characterized for their capability to differentiate toward adipocytes and osteocytes.For adipogenesis, MSCs were incubated for 3 weeks with 5 μg/mL insulin, 10 −6 M dexamethasone, 0.5 mM isobutylmethylxanthine, and 50 μM indomethacin.Then, cells were fixed with 10% formalin, and oil red O staining was used to visualize the accumulation of lipid droplets into the cell vacuoles.For osteogenesis, cultures were fed twice a week for 3 weeks with 10 mM β-glycero-phosphate, 0.2 mM ascorbic acid 2-phosphate, and 10 −8 M dexamethasone.Then, the cells were fixed and extensive mineralization of the extracellular matrix was visualized by alizarin red S.

Substrate Preparation and Cell Culture
For cell seeding, cells were trypsinised and counted.The 2PP-patterned coverglasses (hereon called samples) were washed thoroughly, kept for 12 h in deionised water, disinfected for 12 h in 70% ethanol, washed repeatedly in sterile deionised water, dried and UV-sterilized.Each sample was positioned in a well of Ultra-Low Attachment 24 multi-well plates (Costar 3473, Corning, Corning, NY, USA).The cells were suspended in complete medium and seeded directly in the wells, at a density of 20,000 cells/cm 2 .The cells were incubated for 21 days, with medium freshly replaced every day.The complete medium was made of α-MEM supplemented with 20% FCS, 100 U/mL penicillin G and 0.1 mg/mL streptomycin sulphate.

Morphological Examination
Live cells were imaged in their wells in phase contrast every three days, using a standard microscope (IX70, Olympus, Tokyo, Japan) equipped with a cooled high resolution video camera (4083.CL3, Optika, Ponteranica(BG), Italy).For Scanning Electron Microscopy (SEM), the cells were fixed in the wells in 1.5% glutaraldheide and 0.1 M sodium cacodylate and dehydrated in a graded series of ethanol.The samples were extracted from the wells, air dried, glued onto SEM stubs and gold-coated in a vacuum ion coater.The thickness of the gold coated layer prior to SEM investigation was in the range 15-20 nm.All observations were carried out at 17.5 kV using a SEM (EVO 50 EP, Carl Zeiss, Oberkochen, Germany).

Histological Staining
Samples were fixed with paraformaldehyde 2%.Von Kossa staining was performed to analyze calcium deposits.Samples were incubated in 1% silver nitrate solution under UV ray, washed in 5% sodium tiosolphate, nuclei were countercoloured with fast red, and samples were dehydrated in a graded series of ethanol, ending with xylene.Masson's trichrome stain with alanin blue was used to analyze aspecific collagen content, using a commercial kit (04-010802, BioOptika, Milano, Italy).Safranin staining was performed to analyze glycosaminoglycan (GAG) content.The nuclei were stained with hematoxylin, then samples were incubated in 0.1% Safranin O, and dehydrated in a graded series of ethanol, ending with xylene.Finally, Oil red Owas used to detect lipid content.Samples were firstly stained with a 10 μg/mL solution of Oil red O, then fixed in 4% paraformaldehyde.All samples were mounted on glass slides with Biomount (BioOptika, Milano, Italy) and observed in bright light at 10× and 40×.

Fluorescence Staining and Observation by Confocal Microscopy
For confocal microscopy, the cells were fixed in the wells in paraformaldehyde 2%, permeabilised with Triton 0.2%, blocked with 3% bovine serum albumin (BSA) plus 10% FCS to avoid unspecific labeling, and fluorescently marked.DNA was stained by incubation with 4′,6′-diamidino-2phenylindoledihydrochloride(DAPI)in solution at 10 µg/mL.Actin filaments were marked in green using FITCH-conjugated phalloidin.Cell proliferation was studied by detection of the Ki67 antigen, which is expressed by cells in all the phases of the division cycle, using a red Cy3-conjugated mouse anti-human Ki67 monoclonal antibody (NCL-Ki67-MM1, Novocastra, Nussloch, Germany).Collagen type I, collagen type II and Osteocalcin were immunolabeled with primary antibodies and then marked in red using Cy3-conjugated secondary antibodies.Immunostaining was performed also on expanded MG63 osteosarcoma immortalized cells, for which all the investigated proliferation and differentiation markers are proven to be expressed [14], either using the complete immunostaining protocol (positive control), or using the complete protocol deprived of primary antibody labeling (negative control).Image acquisition and 3D reconstruction were performed at 15× and at 40× by means of a laser confocal microscope (LSM 510 Meta, Carl Zeiss, Oberkochen, Germany).

Estimation of Nuclear Shape
Sequential images, acquired in fluorescence on the DAPI channel, at 1 µm intervals on the Z (vertical) axis, were imported in the Image J 1.43 software (National Institute of Mental Health, Bethesda, MD, USA).The RGB stack sequence was converted into grayscale images (8 bit) and subsequently filtered to reduce noise.The regions of interest corresponding to the cell nuclei and the niche structure were identified and the relevant growing regions were built by segmentation.Solid models of the cell nuclei were reconstructed by numerical interpolation (voxel growing) of subsequent images.The whole analyzed volume was subdivided into three sub-volumes: glass monolayer, niche external wall, and niche internal volume.In each sub-volume, each reconstructed nucleus was associated to its phantom (best fitting) solid ellipsoid.

Statistical Analysis
Five identical experiments were repeated.At each culture time point, cell count, colony count, measurement of colony size and relative distances, were performed on images acquired in fluorescence on the DAPI-labeled samples, in transmission by aninverted microscope (IX70, Olympus, Tokyo, Japan).By this method, all the cell nuclei present on the sample were visualized in projection on the images, instead of what happens in confocal acquisitions where only the projection of a limited sample volume above the cell-populated glass surface is visualized.Colonies were assessed visually on the DAPI-marked cells by the presence of nuclear aggregates.The distance between colonies was assessed by image processing on the DAPI images, by tracing a circle including each visualized colony and by calculating the relative distance between the centers of circle pairs.The cell count was assessed visually on the DAPI-marked cells, bycounting the cell nuclei in square regions of 100 × 100 μm 2 .Cell density was obtained by dividing the cell count of each region by the area of the square region.Results of the cell counts were assigned to experimental groups, based on the count location: (1) uncolonized glass, (2) colonized glass, (3) niche external walls, (4) niche internal volume.The groups were compared using one-way analysis of variance (ANOVA) for independent samples.Pair-wise comparisons among groups were determined with Tukey HSD test, or with Student t-test for independent samples.Differences were considered to be significant if p < 0.05.

Graded Niche Fabrication
We fabricated scaffolds in the SZ2080 photoresist with 1% concentration of Irg photoinitiator.Previously we had fabricated scaffolds in the same SZ2080 photoresist but with Michler's ketone (Bis) photoinitiator [17], which showed strong autofluorescence, hindering the characterization of the fluorescent markers for cell proliferation (Figure 1).The Irg-based photoresist used here was found to be biocompatible [20] similarly to the previously studied Bis-SZ2080, but with greatly reduced auto-fluorescence.These results are in agreement with previous work showing that it is the addition of the photoinitiator which influences both the photosensitivity and fluorescence of the photoresist [18,21].It should also be noted that 2PP is possible in SZ2080 without any photoinitiator,

Potential Clinical Implications
If the observations reported here for animal cells are confirmed for human cells in our future experiments, our "structurally" biomimetic niche system could find potential applications in two main directions.
By extrapolation from our cell density measurements, showing no difference in the cell density occurring in glass colonies with respect to niche colonies at three culture weeks, we can hypothesize that a macroscopic culture plate, completely covered by niches, would induce a homogeneous cell density approaching colony density, i.e., 0.2 million cells/cm 2 , a cell density 67% greater than the average 0.12 million cells/cm 2 measured in standard 2D culture (Figure 6, "average on sample" curve).Thus, one potential application of our niche system could be to produce therapeutic MSC cells in large, pharmaceutically relevant scales.
Secondly, we could exploit the ability of these niches to increase cell renewal while maintaining cell multipotency in the structured layer.MSCs from patients could be expanded and maintained in culture for longer periods, until the formation of niche aggregates, which could be easily harvested from the culture substrate and delivered as a customized cell therapy for chronic disorders, on a repeated basis.The cells with higher "stemness" found within niches could be maintained in culture after aggregate removal, and the process of aggregate fabrication would spontaneously restart production of a further therapeutic cell dose.In our observations, the aggregate cells consistently committed towards the chondrogenic lineage, thus they contained cells potentially useful for cartilage regeneration therapies.

Conclusions
In this work, we studied the response of mesenchymal stem cells over a three-week period in direct laser-written 3D synthetic polymer niches, showing strong proliferation compared to average locations on the sample.By fitting the nuclei of stem cells as ellipsoids, we demonstrated a flatter morphology of stem cells on the outer glass surface and niche outer side walls, whereas inside the niche, stem cells adhered in all spatial directions to the 3D environment and became more round in shape.
We found an average inter-colony distance of 400 μm on flat cover glass, and thus fabricated multiple niches in a hexagonal pattern, spaced by a similar distance.When the scaffolds were closely spaced (~200 μm), resulting in a strong interaction between cells on neighboring niches, this led to the formation of a single, large aggregate on top of the niche ensemble.However, when the distance between the scaffolds was on the same order as the spontaneous separation between colonies (400 μm), colonies were unable to bridge the gap between scaffolds and individual aggregates formed on top of each niche.The average cell density was not dependent on the inter-niche distance.In the hypothesis of using the aggregate cells in a therapeutic application, all the tested inter-niche distances would perform comparably.However, in the hypothesis of therapeutic application of the undifferentiated cells, it would be preferable to obtain large stem cell numbers in short expansion times.In this regard, a shorter inter-niche distance would be preferable.
Over 21 days, such aggregates of stem cells, initially flat and circular colonies, eventually became spherical in shape, both on niches and on the glass substrate, likely recapitulating the contraction phase that precedes the formation of limb drafts in the embryonic connective tissue (i.e., the "mesenchyme").A different stem cell response was observed depending on the environment: a lineage commitment was observed in 2D (cover glass) and 2.5D (niche outer side walls) regions, even without supplements such as Dexamethasone.In the 3D engineered niches, however, we did not observe any evidence of cell differentiation, which is attributed to a reduced deformation of the cell nuclei following an isotropic cytoskeletal tension state.Such an effect could be exploited for the development of novel customized cell therapies.
To give more conclusive evidence of multipotency maintenance within niches, characterization at the gene expression level of cells inside and outside the niches is required, which was not possible on the relatively few cells (140 per sample, on average) contained inside the seven niches of each substrate in this study.To this purpose, we are now extending the culture surface covered by the niches to larger areas up to 1 cm 2 , with the aim of obtaining larger niche-cultured cell numbers compared to the average 17 cells/niche available now.In addition, we are currently performing experiments for functionalizing the niche surface with respect to its stiffness by a suitable coating, to further control cell adhesion.

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