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1

Microfabrication techniques and surface functionalisation for the culture of neural stem cells

100%
Rossi F., Colpo P., Mehn D., Nowak J., Ruiz A.
Acta Neurobiologiae Experimentalis
|
2011
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tom 71
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nr S
We present different processes used for the patterning and growth of stem cells based on microspotting, microcontact printing and 3D patterning. Microspotting and microcontact-printing technique have been performed to produce micropatterned surfaces for cell-biological applications. Biomolecules have been micropsotted and microstamped on plasma-polymerized poly ethylene glycol substrates. The patterns exhibited a firm stability and an improved feasibility for controlling cell localization, proliferation and even differentiation. Production and application of 3D substrate by combination of lithography and in situ UV cross linking of photoresist are also detailed. An application of these processes is the biofunctional surfaces were incubated with suspensions of human umbilical cord blood neural stem cells (HUCB-NSC). It was clearly observed how the cells adhered and grew in the protein patterned regions. After 4 weeks of culturing the cells were still anchored in the patterns. Immunocytochemistry studies indicate that cell differentiation can be controled by the combination of interface engineering and culture conditions.
2
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Determination of mechanical properties for wood pellets used in DEM simulations

86%
Gallego E., Fuentes J. M., Ruiz A., Hernandez-Rodrigo G., Aguado P., Ayuga F.
International Agrophysics
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2020
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tom 34
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nr 4
3

Patterning of human cord blood-derived stem cells on single cell posts and lines: implications for neural commitment

86%
Zychowicz M., Mehn D., Ruiz A., Frontczak-Baniewicz M., Rossi F., Buzanska L.
Acta Neurobiologiae Experimentalis
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2012
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tom 72
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nr 4
Using stem cells (SC) in new strategies for clinical treatment requires control of stem cell fate decision and the ability to govern their patterning and commitment in tissue engineering. Neural stem cells and other adult SC can respond to the different components of the microenvironment and their spatial arrangement in the stem cell niche. It has been shown previously by our group that different composition and architecture of patterned bioactive domains influence the developmental response of neural stem cells. In the present report we verify the commitment and differentiation of neural stem cells derived from human cord blood (HUCB-NSC) by a single cell patterning system. Microcontact printing technology was used to generate single cell positioning areas of different geometry: 10 ^m-thin lines and 10 ^m-width one cell posts. The commitment and differentiation of HUCB-NSC cells cultured on different surfaces were dependent on the geometry and the type of biomateriał present in bioactive domains. Fibronectin promoted neuronal protrusion outgrowth (P-tubulin III and MAP-2 positive cells) and gap junction development (Cx43 marker) between cells growing on lines while poly-L-lysine promoted HUCB-NSC differentiation into astrocytic, glial fibrillary acidic protein expressing (GFAP positive) cell phenotype. Here we also demonstrate by scanning electron microscopy that morphology of cells on the patterned surface is highly dependent upon the type of biomolecules used for printing. These kinds of platforms can be used for investigating the influence of spatial organization of environment on the SC fate decision and for studying the molecular processes occurring in a single cell.
4

Controlling of HUCB-NSC culture by micropatterned biofunctionized surfaces

86%
Zychowicz M., Buzanska L., Ruiz A., Coecke S., Rossi F., Domanska-Janik K.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
Stem cells have potential to maintain in organism by self-renewal division and ability to multilineage differentiation. Cell-cell contacts, paracrine signals and extracellular matrix proteins occurring in the neurogenic niches are main determinants of neural stem cells fate and dynamic of their differentiation. Aim of this study is to investigate whether could nano/micro-patterned, biofunctionalized surface guides human cord blood derived neural stem cell to growth and differentiation. Methods: To investigate the infl uence of the cell plating density we used the microcontact printed patterns of adhesive substrate (poly-L-lysine) on cell-repellent poly-ethylene glycol (PEG) substrate. Two different geometries of the patterning have been applied: to test proliferative response the cells were seeded in different densities on the pattern with separated pitches, while to verify their ability to differentiation the culture medium was supplemented with cAMP and cells seeded on the surface patterned with interconnecting lines. After 2, 4 and 7 days the cells were fi xed and immunostained for Ki67 (proliferation marker) and beta III tubulin/ S100β markers for neuronal/astrocytic lineage. Results: The low cell density of HUCB-NSC (104 cell/cm2 ) and the presence of neuromorphogenes (cAMP) supports neural stem cell differentiation, while enhanced initial cell density promotes the growth rate (increase of the cell number falling on biofunctionalized unit per 100 μm2 surface). Conclusions: Micropatterned platforms with biofunctionalized surface can be used for screening of the plethora of extracellular signals directing neural stem cell to growth and differentiation.
5

Proliferation capacity of cord blood derived neural stem cell line on different micro-scale biofunctional domains

72%
Zychowicz M., Mehn D., Ruiz A., Colpo P., Rossi F., Frontczak-Baniewicz M., Domanska-Janik K., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr 1
Physical interactions of cells with the adhesive substrates of the microenvironment as well as the presence of the soluble growth factors are important for the proliferation capacity of neural stem cells. We have used biofunctionalized surface domains microcontact printed with either synthetic polyaminoacid poly-L-lysine or extracellular matrix (ECM) component such as fibronectin, to study the proliferation capacity of human umbilical cord blood-derived neural stem cells (HUCB- NSC). The proliferation measured by the expression of Ki-67 protein was accompanied by the investigation of the cell morphology under the transmission and scanning electron microscopy in different culture time, plating densities of cells and medium condition (serum-free or 2% of FBS). The poly-L-lysine domains of defined micro-scale area promoted the presence of round, loosely attached Ki-67-positive cells, while fibronectin domains of the same size allowed appearance of flattened, strongly attached cells with more differentiated phenotype. These results were in agreement with the non-specific, electrostatic type of interaction between cell and substrate on poly-L-lysine and integrin receptor-mediated specific adhesion on fibronectin. In this report we have described in vitro culture conditions, which allow for immobilization of the non- differentiated and highly proliferating population of neural stem/progenitor cells to the biofunctionalized surface. The microarrays with bioactive domains allocating non-differentiated and proliferating neural stem/progenitor cells may find application for drug and chemicals toxicology screening of diverse factors influencing neural development.
6

Human cord blood cells in neurotoxicology: Advantage of emerging technologies

72%
Buzanska L., Zychowicz M., Ruiz A., Ceriotti L., Sobanski T., Rossi F., Calpo P., Domanska-Janik K., Coecke S.
Acta Neurobiologiae Experimentalis
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2009
|
tom 69
|
nr 1
Stem cell technology provides a new tool for better understanding the mechanisms involved in compound-induced adverse reactions of the organism, which particularly applies to the fi eld of developmental neurotoxicity. Human Umbilical Cord Blood Neural Stem Cell (HUCB-NSC) line is a model system where key neurodevelopmental processes were investigated by conventional and emerging techniques. The advantage of the HUCB-NSC line is that cells are of human origin, non-transformed and can be cultured/harvested at different developmental stages. In this report emerging nano/micro-technologies were used to create biofunctional micropatterns and multi electrode array chips for the detection of cell behaviour and vulnerability to toxic compounds. Micropatterned surfaces were produced by a spatial arrangement of different functional domains. This included a nano/micro-fabrication technique like contact printing in order to create a pattern of separated or interconnected polypeptide spots directing cell growth and differentiation. Another approach was to create protein microarrays by piezoelectric (non contact) deposition of extracellular matrix proteins. Such a method allows defi ned active areas to be produced on the same platform and enables unambiguous access to cell behavior on different protein types and concentrations. HUCB-NSC were shown to adhere and differentiate on microarray platforms in a protein type, concentration and cell density dependent manner. Sensor and omics techniques applied to HUCB-NSC included measurements of electrical activity using multielectrode array chips and metabolite profi ling by mass spectrometry. Spontaneous electrical fi eld potentials and the protein composition of tested cells were sensitive to neurotoxic treatments in a developmental stage specifi c manner.
7

Patterned growth and differentiation of human cord blood-derived neural stem cells on bio-functionalized surfaces

72%
Buzanska L., Ruiz A., Zychowicz M., Rauscher H., Ceriotti L., Rossi F., Colpo P., Domanska-Janik K., Coecke S.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 1
Bio-functionalized surfaces were prepared to study the adherence and differentiation capacity of neural stem cells derived from human umbilical cord blood (HUCB-NSC). Cell growth platforms containing arranged arrays of adhesive molecules were created by microcontact printing on a biologically inert surface. Biomolecules used to prepare microarray platforms included the extracellular matrix protein fibronectin and the polyaminoacid poly-L-lysine. HUCB-NSC plated on microplatforms at various serum conditions showed serum and molecule type dependent capacity for adhesion and differentiation. Poly-L-lysine allowed the maintenance of stem-like non differentiated cells attached to the surface, whereas fibronectin promoted spreading and neural commitment. Serum deprivation did not influence the attachment of HUCB-NSC to fibronectin, but significantly enhanced the attachment to poly-L-lysine and promoted dBcAMP induced neuronal differentiation. A bio-pattern of squares with interconnecting lines was used to guide neuronal differentiation by directing cell protrusion outgrowth. Tailoring the geometry of the bio-pattern enabled directing and monitoring of the neural stem cells development in the large scale multiparameter biotests.
8

HUCB-NSCS growth control by biofunctionalized surfaces

59%
Zychowicz M., Ruiz A., Cerioti L., Lisboa P., Mehn D., Coecke S., Rausher H., Colpo P., Rossi F., Domanska-Janik K., Buzanska L.
Acta Neurobiologiae Experimentalis
|
2009
|
tom 69
|
nr 3
Cell growth platforms with biofunctionalized surfaces were fabricated to control and direct HUCB-NSC fate decisions. Two different nano/micro techniques: microcontact printing and piezoelectric non-contact spotting were used to allocate biomolecules (poly-Llysine and fi bronectin) on cell-repellent, non adhesive substrate. Such methods allow controlling the spatial distribution and content of the biomolecules on the microarray and governing cell adhesion in unspecifi c (electrostatic) or specifi c (receptor-mediated) manner. Patterning of biomolecules in different conditions on the single growth platforms enables to infl uence and compare stem cells developmental processes (proliferation /differentiation) at variable environments. To refl ect/mimic stem cell niche we applied functional domains containing ECM protein spotted together with the small signaling molecules (notch, wnt, shh). Such approach enable directing of neural stem cell developmental program by inducing intracellular molecular pathways leading to either self renewing- or differentiating- (neuronal or astrocytic) state. We characterized active biofunctionalized domains on fabricated microarrays by applying two methods of surface analysis: ellipsometry measurement and surface plasmon resonance system. This type of bioengineered cell growth platforms can be used for screening the mechanisms governing neural stem cell fate decisions and adverse reactions upon environmental stimuli. Grant No 0141/B/ P01/2008/35.
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