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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/069—Vascular Endothelial cells
  • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0661—Smooth muscle cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/70—Polysaccharides
  • C12N2533/74—Alginate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
  • C12N2537/10—Cross-linking

Definitions

• the invention relates to an artificial hollow cell microfiber having a structure, histology and mechanical properties similar to those of vessels of the animal vascular system.
• the invention also relates to a manufacturing method for obtaining such a hollow cellular microfiber.
• the invention finds applications particularly in the field of tissue engineering and tissue grafts, to allow the vascularization of tissues, and in the pharmacological field, for the study in particular of candidate molecules having activity related to vascularization.
• tissue engineering has been developed, with the aim of artificially recreating blood or lymphatic vessels, in particular to allow tissue vascularization in vitro.
• one method is to mold a cell-loaded hydrogel around agarose-based tubes.
• the agarose tubes are subsequently removed to create microtube networks (Bertassoni et al., Lab Chip 2014 Jul 7; 14 (13): 2202-2211).
• Another technique is to flow a collagen gel onto a gelatin or polydimethylsiloxane (PDMS) tube, which is removed once the gelled collagen matrix (Backer et al., Lab Chip. 2013 Aug 21; 13 (16): 3246 -3252 and Jimenez-Torres et al., Mol Mol Methods 2016, 1458: 59-69).
• PDMS polydimethylsiloxane
• microfibers containing endothelial cells wrapped in a hydrogel layer have been obtained by coextrusion (Onoe et al., Nature Materials 31 March 2013). However, these microfibers do not have mechanical properties comparable to those of the blood or lymphatic vessels.
• the inventors have discovered that it is possible to manufacture hollow cell microfibers reproducing histologically and mechanically vessels of the mammalian vascular system, such as blood vessels. . More specifically, the inventors have developed a method of encapsulation of endothelial cells and smooth muscle cells in an alginate envelope, inside which the cells organize themselves in homocentric layers around the skin. 'a light.
• the method according to the invention makes it possible to obtain tubes of lengths and diameters that can be modulated according to the needs. In particular, it is possible to produce tubes of a few centimeters and up to more than 1 meter.
• the outer diameter of the tubes according to the invention may vary from 70 ⁇ to more than 5 mm, so as to mimic any type of blood and lymphatic vessels, from the veins to the arteries.
• the light extends throughout the length of the tube, making said tubes perfusable. The vessels thus obtained can be easily individualized and manipulated.
• the subject of the invention is therefore an artificial hollow cellular microfiber comprising, successively, organized around a light
• At least one layer of smooth muscle cells at least one layer of smooth muscle cells; an extracellular matrix layer; and optionally
• the cellular microfiber is a blood vessel or lymphatic vessel.
• the invention also relates to a process for preparing such a hollow cellular microfiber, according to which a hydrogel solution and a solution of cells comprising endothelial cells and smooth muscle cells in an extracellular matrix are coextruded concentric in a crosslinking solution capable of crosslinking at least one polymer of the hydrogel solution.
• FIG. 1 Cross-sectional representation of a hollow cellular microfiber according to an exemplary embodiment of the invention, comprising successively, from the outside towards the inside, an alginate outer layer (1), an extracellular matrix layer ( 2), a layer of smooth muscle cells (3), a layer of endothelial cells (4) and a central lumen (5);
• FIG. 2 Schematic representation of a concentric coextrusion system that can be used to produce the cellular microfibers according to the invention, wherein a first pump comprises an alginate solution (ALG), a second pump comprising an intermediate solution comprising sorbitol (SI), and the third pump comprising a solution of cells (C), these three solutions being brought to a coextrusion tip and the tip (6) being immersed in a crosslinking bath (7) to form the cellular microfiber hollow (8);
• ALG alginate solution
• SI intermediate solution comprising sorbitol
• C solution of cells
• Figure 3 Microscope views of the tubular structure of a cellular microfiber obtained according to the method of the invention.
• the cells are round and disposed within the entire alginate tube;
• the cells are anchored on the internal edges of the alginate tube, via the extracellular matrix, to form a lumen inside the tube;
• Figure 4 Study of the impact of the co-extrusion rates of an alginate solution (a), a solution of sorbitol (s) and a solution of cells (c) on the thickness of the outer layer of alginate in the hollow cell microfibers obtained;
• FIG. 5 Study of the external (EXT) and internal (INT) diameters of various hollow cellular microfibers obtained according to the method of the invention, as a function of the diameter of the coextrusion outlet tip (abscissa axis: 300 ⁇ , 350 ⁇ , 450 ⁇ );
• Figure 6 View of an empty alginate tube 900 ⁇ diameter, obtained by extrusion with an outlet nozzle diameter 900 ⁇ ;
• Figure 7 Study of the contraction of hollow cellular microfibers according to the invention in the presence of Endotheline 1 (ET1);
• Figure 8 Study of the increase in intracellular calcium concentration (l // uo ) in endothelial cells of the human umbilical cord vein (HUVEC) and in the smooth muscle cells (SMC) of hollow cellular microspheres over time , under the effect of endothelin 1.
• the invention relates to artificial hollow cell microfibers, the histology and mechanical and physiological properties of which mimic those of vessels of the animal vascular system, and in particular of the mammalian vascular system.
• microfibers based on smooth muscle cells and endothelial cells, whose organization in concentric layers around a light renders said microfibers perfusable.
• perfusable is meant in the context of the invention that it is possible to inject a fluid into said microfiber, within which it can circulate.
• the hollow cellular microfibers according to the invention are also impervious in that the fluid injected into said microfibers does not escape or only very slightly through the thickness of the microfibres.
• the tightness of a microfiber according to the invention depends mainly on the degree of confluence of the cells in said microfiber. The degree of confluence can in particular be adapted by varying the number of cells injected during the formation of the microfiber.
• the microfibers according to the invention can be manipulated because they are individualized.
• the cellular microfiber is a hollow tubular structure, containing substantially homocentric layers, in that they are organized successively around the same point.
• the central lumen 5 of the microfiber is bordered by the endothelial cell layer 4, which is surrounded by the smooth muscle cell layer 3, itself surrounded by an extracellular matrix layer 2 and optionally an outer layer of hydrogel 1 (figure 1).
• a cross section of the cellular microfiber according to the invention thus comprises successive substantially concentric layers.
• the light is generated, at the time of tube formation, by the smooth and endothelial muscle cells that self-assemble and orient spontaneously with respect to the extracellular matrix.
• the light contains a liquid and more particularly the culture medium.
• the hollow cellular microfiber comprises an outer layer of hydrogel.
• the "outer layer of hydrogel” designates a three-dimensional structure formed from a matrix of polymer chains swollen with a liquid, and preferably water.
• the polymer or polymers of the outer layer of hydrogel are crosslinkable polymers when subjected to a stimulus, such as a temperature, a pH, ions, etc.
• the hydrogel used is biocompatible, in that it is not toxic to the cells.
• the hydrogel layer must allow the diffusion of oxygen and nutrients to feed the cells contained in the microfiber and allow their survival.
• the polymers of the hydrogel layer may be of natural or synthetic origin.
• the outer layer of hydrogel contains one or more polymers among sulfonate-based polymers, such as sodium polystyrene sulfonate, acrylate-based polymers, such as sodium polyacrylate, polyethylene glycol diacrylate, the gelatin methacrylate compound, polysaccharides, and especially polysaccharides of bacterial origin, such as gellan gum, or of plant origin, such as pectin or alginate.
• the outer hydrogel layer comprises at least one of alginate.
• the outer layer of hydrogel comprises only alginate.
• alginate is understood to mean linear polysaccharides formed from ⁇ -D-mannuronate (M) and ⁇ -L-guluronate (G), salts and derivatives thereof.
• the alginate is a sodium alginate, composed of more than 80% of G and less than 20% of M, with an average molecular mass of 100 to 400 kDa (for example: PRONOVA ® SLG100) and a total concentration of between 0.5% and 5% by weight (weight / volume).
• the outer layer of hydrogel can strengthen the rigidity of the cellular microfiber and thus facilitate its handling.
• the hydrogel layer comprises polymers capable of limiting cell adhesion ("cell-repellent”), in order to facilitate, if necessary, the separation of said hydrogel layer from the cellular microfiber or its degradation without affecting the structure of the cellular microfiber.
• cell-repellent polymers capable of limiting cell adhesion
• the cellular microfiber is devoid of an outer hydrogel layer and directly comprises, as the outermost layer, an extracellular matrix layer.
• the extracellular matrix layer forms a gel on the inner face of the hydrogel layer, that is to say the face directed towards the light of the microcompartment.
• the extracellular matrix layer comprises a mixture of proteins and extracellular compounds required for cell culture.
• the extracellular matrix comprises structural proteins, such as laminins containing the subunits ⁇ 1, ⁇ 4 or ⁇ 5, the subunits ⁇ or ⁇ 2, and the subunits ⁇ or ⁇ 3, vitronectin, laminins, collagen, as well as growth factors, such as TGF-beta and / or EGF.
• the extracellular matrix layer consists of or contains Matrigel ®, the Geltrex ®, collagen, including type 1 collagen to 19, modified or not, gelatin, fibrin, hyaluronic acid, chitosan, or a mixture of at least two of these components.
• the cellular microfiber comprises smooth muscle cells, organized in one or more layers around and possibly at least partly in the extracellular matrix layer.
• the smooth muscle cells can be chosen in particular from vascular smooth muscle cells, smooth muscle cells, smooth muscle cells of the digestive tract, bronchial smooth muscle cells, smooth muscle cells of the kidneys, smooth muscle cells of the bladder, dermal smooth muscle cells, smooth muscle cells of the uterus and smooth muscle cells of the eyeball, mammalian and especially human.
• the smooth muscle cells are selected from smooth muscle cells of lymphatic or vascular origin, such as smooth muscle cells of umbilical artery, smooth muscle cells of coronary artery, smooth muscle cells of pulmonary artery, etc. .
• the smooth muscle cells are coronary artery smooth muscle cells, such as smooth muscle cells of the human coronary artery.
• smooth muscle cells are obtained from pluripotent induced stem cells, which have been forced to differentiate into smooth muscle cells.
• the thickness of the smooth muscle cell layer (s) may vary depending on the destination of the cellular microfiber. "Thickness” means the dimension in a cross section of the microfiber extending radially from the center of said cross section. Smooth muscle cells allow the contraction of the microfiber. It is therefore possible to adapt the contractile force of the cellular microfiber, depending on whether it is intended to be used as a blood vessel or lymphatic vessel, but also according to the nature of said reproduced vessel (artery, vena cava, vein). , venule, etc.). The person skilled in the art knows what is the expected contractile force as a function of the vessel to be reproduced, and thus knows how to adapt the thickness of the layer (s) of the smooth muscle layers, as well as the nature of the smooth muscle cells.
• the layer or layers of smooth muscle cells contains at least 95% by volume, preferably at least 96%, 97%, 98%, 99% of smooth muscle cells and matrix produced by said cells.
• the layer or layers of smooth muscle cells may optionally include endothelial cells.
• the volume percentage of endothelial cells in the smooth muscle cell layer is less than 5%, preferably less than 4%, 3%, 2%, 1%.
• the hollow cellular microfiber comprises a layer of endothelial cells, bordering and delimiting the central lumen.
• the endothelial cells may be chosen from umbilical cord vein endothelial cells (UVEC), skin microvessel endothelial cells (DMEC), dermal blood endothelial cells (DBEC), dermal endothelial cells (DLEC), endothelial cells of heart microvessels (CMEC), endothelial cells of lung microvessels (PMEC) and endothelial cells of uterine microvessels (UtMEC), mammalian and especially human.
• UVEC umbilical cord vein endothelial cells
• DMEC skin microvessel endothelial cells
• DBEC dermal blood endothelial cells
• DLEC dermal endothelial cells
• CMEC endothelial cells of heart microvessels
• PMEC endothelial cells of lung microvessels
• UtMEC endo
• the endothelial cells are endothelial cells of the umbilical cord vein (UVEC), including endothelial cells of the human umbilical cord vein (HUVEC).
• UVEC umbilical cord vein
• HAVEC human umbilical cord vein
• endothelial cells are obtained from pluripotent induced stem cells, which have been forced to differentiate into endothelial cells.
• the cellular microfiber comprises a single layer of endothelial cells.
• the endothelial cell layer or layers contain at least 95% by volume, preferably at least 96%, 97%, 98%, 99% endothelial cells and matrix produced by said cells.
• the layer or layers of endothelial cells can possibly include smooth muscle cells.
• the volume percentage of smooth muscle cells in the endothelial cell layer is less than 5%, preferably less than 4%, 3%, 2%, 1%.
• the cells used to produce the cellular microfiber according to the invention are human cells.
• the ratio of average endothelial cells / smooth muscle cells, in cm 2 , in a hollow cellular microfiber of the invention is between 3/1 and 2/1.
• the internal diameter of the cellular microfiber is between 50 ⁇ and 500 ⁇ , preferably between 50 ⁇ and 200 ⁇ , more preferably between 50 ⁇ and 150 ⁇ , still more preferably between 50 ⁇ and 100 ⁇ , +/- 10 ⁇ .
• internal diameter is meant the diameter of the light of the microfiber.
• the internal diameter of the cellular microfiber is 100 ⁇ .
• the internal diameter is 70 ⁇ .
• the outer diameter of the cellular microfiber may also vary.
• outer diameter is meant the largest diameter of the microfiber.
• the outer diameter is advantageously between 250 ⁇ and 5 mm.
• the outer diameter is advantageously between 70 ⁇ and 5 mm, preferably between 70 ⁇ and 500 ⁇ , more preferably between 70 ⁇ and 200 ⁇ , even more preferably between 70 ⁇ and 150 ⁇ . , +/- 10 ⁇ .
• the outer diameter of the microfiber, in the presence of the outer layer of hydrogel is 300 ⁇ .
• the outer diameter of the microfiber, in the absence of the outer layer of hydrogel is 150 ⁇ .
• the cellular microfiber according to the invention comprises an outer layer of hydrogel of 100 to 150 ⁇ in thickness, a thickness of cells (endothelial cells and smooth muscle cells) of 150 to 200 ⁇ and a light from 100 to 150 ⁇ in diameter.
• the cellular microfiber according to the invention has a length, or greater dimension, of at least 50 cm, preferably at least 60 cm, 70 cm, 80 cm, 90 cm, 100 cm, 110 cm, or more.
• the invention also relates to a process for preparing a hollow cellular microfiber according to the invention. More particularly, the invention proposes to encapsulate endothelial cells and smooth muscle cells in an outer hydrogel shell within which said cells will rearrange to form substantially concentric layers and provide a central lumen. The encapsulation is done by means of a concentric coextrusion process, in which the hydrogel solution is coextruded with the cell solution directly in a crosslinking bath, or crosslinking solution, comprising a crosslinking agent for crosslinking the hydrogel and thus form the outer shell around the cells.
• any extrusion process for concentrically coextruding hydrogel and cells can be used.
• the method according to the invention is implemented by means of an extrusion device with double or triple concentric envelopes as described in patent FR2986165.
• crosslinking solution means a solution comprising at least one crosslinking agent adapted to crosslink a hydrogel comprising at least one hydrophilic polymer, such as alginate, when it is applied. contact with it.
• the crosslinking solution may for example be a solution comprising at least one divalent cation.
• the crosslinking solution may also be a solution comprising another known crosslinking agent of the alginate or of the hydrophilic polymer to be crosslinked, or a solvent, for example water or an alcohol, adapted to allow crosslinking by irradiation or by any other means. other technique known in the art.
• the crosslinking solution is a solution comprising at least one divalent cation.
• the divalent cation is a cation which makes it possible to crosslink alginate in solution, it may be, for example, a divalent cation chosen from the group. comprising Ca 2+ , Mg 2+ , Ba 2+ and 5r 2+ , or a mixture of at least two of these divalent cations.
• the divalent cation, for example Ca 2+ may be combined with a counter-ion to form, for example, solutions of the CaC or CaCOs type, which are well known to those skilled in the art.
• the crosslinking solution may also be a solution comprising CaCO3 coupled to Glucono delta-lactone (GDL) forming a CaCOs-GDL solution.
• the crosslinking solution can also be a mixture of CaCOs-CaSC GDL.
• the crosslinking solution is a solution comprising calcium, in particular in the Ca 2+ form,
• the divalent cation concentration in the crosslinking solution is between 10 and 1000 mM.
• the crosslinking solution may comprise other constituents, which are well known to those skilled in the art, than those described above, in order to improve the crosslinking of the hydrogel sheath under the conditions, in particular time and / or temperature, special.
• the endothelial cells have previously been cultured in a culture medium comprising vascular endothelial growth factors (VEGF) so as to promote the formation of endothelium and angiogenesis.
• VEGF vascular endothelial growth factors
• the endothelial cells were previously cultured in EGM-2 ® medium.
• the smooth muscle cells have been previously cultured in a culture medium comprising growth factors adapted to the culture of smooth muscle cells, such as transforming growth factor ⁇ , EGF factor, bFGF factor, etc.
• growth factors adapted to the culture of smooth muscle cells such as transforming growth factor ⁇ , EGF factor, bFGF factor, etc.
• smooth muscle cells have been previously cultured in medium SmGM2 ® from Lonza or in a culture medium specifically adapted to the smooth muscle cells marketed by Promoceli society (eg the middle HCASMC ®, HAoSMC ® , etc.),
• the cell solution used for coextrusion comprises endothelial cells and smooth muscle cells suspended in extracellular matrix.
• the cell solution comprises between 20 and 30% by volume of cells and between 70 and 80% by volume of extracellular matrix.
• the volume ratio of endothelial cells / smooth muscle cells in the cell solution is advantageously between 3/1 and 2/1.
• the coextrusion is carried out so that the hydrogel solution surrounds the cell solution.
• coextrusion also involves an intermediate solution, including sorbitol.
• the coextrusion is carried out so that the intermediate solution is disposed between the hydrogel solution and the cell solution (FIG. 2A).
• the extrusion rate of the alginate solution is between 1 and 10 ml / h, preferably between 2 and 5 ml / h, even more preferably equal to 3 ml / h, and preferred way equal to 2 ml / h, +/- 0.5 ml / h.
• the extrusion rate of the intermediate solution is between 0.1 and 5 ml / h, preferably between 0.5 and 1 ml / h, more preferably equal to 0.5 ml. / h, +/- 0.05 ml / h.
• the extrusion rate of the cell solution is between 0.1 and 5 ml / h, preferably between 0.5 and 1 ml / h, more preferably still equal to 0.5. ml / h, +/- 0.05 ml / h.
• the coextrusion speed of the different solutions can be easily modulated by those skilled in the art, so as to adapt the internal diameter of the microfiber and the thickness of the hydrogel layer.
• the extrusion rate of the hydrogel solution is greater than the extrusion rate of the cell solution and optionally of the intermediate solution.
• the extrusion rate of the hydrogel solution is at least two, three, or four times greater than the extrusion rate of the cell solution.
• the extrusion rates of the cell solution and the intermediate solution are identical.
• the extrusion rate of the hydrogel solution is 2 ml / h, +/- 0.05 ml / h, and the extrusion speed of the cell solution as the intermediate solution is 0.5 ml / h, +/- 0.05 ml / h.
• the extrusion rate of the hydrogel solution is 9 ml / h, +/- 0.05 ml / h, and the extrusion rate of the cell solution as the intermediate solution is 3 ml / h, +/- 0.05 ml / h.
• the extrusion rate of the hydrogel solution is 3 ml / h, +/- 0.05 ml / h
• the extrusion speed of the cell solution is 2 ml / h
• the extrusion rate of the intermediate solution is 1 ml / h, +/- 0.05 ml / h.
• the extrusion rate of the hydrogel solution is 2 ml / h, +/- 0.05 ml / h, and the coextrusion rate of the cell solution as the intermediate solution is 0.5 ml / h, +/- 0.05 ml / h.
• the extrusion rate of the hydrogel solution is 2 ml / h
• the coextrusion rate of the cell solution as the intermediate solution is In another particular embodiment of the process according to the invention, the extrusion rate of the hydrogel solution is 2 ml / h, +/- 0.05 ml / h
• the extrusion rate of the cell solution is 0.5 ml / h, +/- 0.05 ml / h
• the extrusion rate of the intermediate solution is 1.5 ml / h, +/- - 0.05 ml / h.
• the extrusion rate of the hydrogel solution is 2 ml / h, +/- 0.05 ml / h
• the extrusion speed of the cell solution is 1.5 ml / h
• the extrusion rate of the intermediate solution is 0.5 ml / h, +/- 0.05 ml / h.
• the reticuiation solution, the intermediate solution and the cell solution are loaded into three concentric compartments of a coextrusion device. so that the crosslinking solution (ALG), forming the first stream, surrounds the intermediate solution (SI) which forms the second stream, which itself surrounds the cell solution (C) which forms the third stream.
• the first flow is the rigid outer shell of hydrogel.
• the second stream constitutes the intermediate envelope and the third flows the inner envelope containing the cells.
• the method according to the invention makes it possible to encapsulate smooth muscle cells and endothelial cells in an external hydrogel sheath.
• the inventors have observed that after only a few hours, the cells contained in this hydrogel sheath reorganize themselves, so that the endothelial cells delimit a longitudinal internal lumen extending over the entire length of the cellular microfiber, and that the smooth muscle cells are oriented outwards with respect to the light.
• the presence of extracellular matrix during coextrusion seems necessary for the cells to become anchored to the matrix and thus spread, divide and proliferate.
• the matrix also makes it possible to reduce the risks of apoptosis of the cells inside the cellular microfiber, and promotes the phenomenon of cellular reorganization within the hydrogel sheath.
• the cellular microfiber obtained by coextrusion is maintained in a suitable culture medium for at least 10 h, preferably at least 20 h, even more preferably at least 24 h before being used.
• This latency advantageously allows the cells to reorganize in the hydrogel sheath so as to form the concentric layers around a light, as described above.
• the hollow cellular microfiber obtained by coextrusion that is to say a microfiber comprising a hydrogel sheath, or to proceed with hydrolysis of said sheath in order to recover a microfiber free of hydrogel.
• the hollow cell microfibers that are the subject of the present invention can be used for many applications, in particular for medical or pharmacological purposes.
• the cellular microfibers according to the invention can in particular be used for identification and / or validation tests of candidate molecules having an action on all or part of the vascular system, and in particular on the blood or lymphatic vessels.
• such microfibers can be used to test the anti-angiogenic, anti-thrombotic, blood pressure regulating, blood gas transporting, etc., properties of candidate molecules.
• the hollow cell microfibers according to the invention can also be used in tissue engineering, in order to vascularize samples of synthetic biological tissues and thus increase their viability.
• tissue engineering in order to vascularize samples of synthetic biological tissues and thus increase their viability.
• vascularized tissue samples can be used for example by the pharmaceutical and cosmetics industries, in order to carry out in vitro tests, in particular as an alternative to animal testing.
• hollow cell microfibers according to the invention can be used in regenerative medicine, in order to allow the vascularization of synthetic organs, such as skin, cornea, liver tissue, etc. obtained by 3D printing or other, before grafting them into a subject.
• Human umbilical cord endothelial cells (HUVEC) cultured in culture medium comprising passage VEGF 3 (p3), 4 (p4) or 5 (p5), supplied cryopreserved in -80 liquid nitrogen ° C under the reference c-12205 by the company PromoCell ® .
• Human coronary artery smooth muscle cells in passage 2 (p2), supplied in the cryopreserved state in liquid nitrogen at -80 ° C. under the reference CC-2583 by the company Lonza.
• Endothelial cell freezing medium Cryo-SFM from PromoCell (ref C-29912).
• Smooth muscle cell culture medium SmGm2-bullet ® kit from the company Lonza (ref CC-3182) (medium at + 4 ° C and supplements at -20 ° C).
• Medium for detaching smooth muscle cells Detach KIT ® from PromoCell Company (ref C-41210).
• Freezing medium for smooth muscle cells Cryo-SFM from PromoCell (ref C-29912). Solutions:
• Hydrogel Solution 2.5% Alginate w / v (LF200FTS) in 0.5mM SDS
• Extracellular matrix Matrigel ® classic (without phenol red and with growth factors) Treatment of HUVEC endothelial cells: Amplification
• HUVEC cells at stage p3 are thawed and amplified according to the usual protocols up to stage p5, p6 or p7, the coextrusion being carried out with cells between stages p5 and p7.
• SMC cells at stage p2 are thawed and then cultured according to the usual protocols up to stage p5, p6 or p7, the coextrusion being carried out with cells between stages p5 and p7.
• Coextrusion system
• Coextrusion of the three solutions in a solution of Ca 2+ as described above made it possible to obtain tubes, or cellular hollow microfibers, of approximately 1 meter in length and with an external diameter of 300 ⁇ .
• the cells After 24 hours (FIG. 3B), the cells reorganized and self-assembled inside the alginate tube so as to create a central lumen with a diameter of approximately 150 ⁇ .
• the tube then comprises successively, and organized concentrically around the light, a layer of HUVEC cells, an SMC cell layer, a Matrigel ® layer and a crosslinked alginate layer.
• Example 1 The hollow cell microfibers obtained in Example 1 were characterized by means of specific markers by immunofluorescence and confocal microscopy. The reorganization of the cells within the alginate envelope was followed by videomicroscopy.
• the cellular microfibers, or tubes, were fixed at different times (J1 / J5), with 4% paraformaldehyde diluted in DMEM without phenol red (PAN), overnight at 4 ° C.
• the cells of the tubes were then permeabilized (30 min in 1% triton in DMEM without phenol red, at room temperature with stirring). Non-specific sites of cells were saturated for one hour at 4 ° C in BSA1% / SVF2% solution (bovine serum albumin and fetal calf serum).
• the cellular microfibers were then placed in the presence of specific primary antibodies, each directed against a protein of interest: - CD31: endothelial cell membrane specific marker
• aSMA alpha smooth muscle actin
• VE-Cadherin a specific marker for endothelial cell junctions and the formation of an impermeable endothelium
• KI67 Specific marker of cell proliferation
• aCaspase3 Specific marker of apoptosis.
• the primary antibody was diluted to l / 100th in DMEM without phenol red + 1% BSA / 2% FCS overnight under stirring at 4 ° C. After 2x15 min of washing in DMEM without phenol red, the tubes were incubated with a secondary antibody (which will specifically recognize the primary antibody) coupled to a fluorochrome, diluted to 1/1000 in DMEM without red phenol + 1% BSA. / 2% FCS for 1h at room temperature. After 2x15 min washes in DMEM without red phenol, the tubes were analyzed by confocal microscopy to visualize the fluorescence. Results:
• SMC specific aSMA marker, alpha Smooth Muscle Actin
• HUVEC specific CD31 marker
• microfibers The perfusability of the microfibers was also evaluated by connecting them to an injection system comprising fluorescent solutions.
• An infusion system for hollow cellular microfibers has been developed using Pasteur glass pipettes drawn under the flame to a diameter corresponding to the internal diameter of the cellular microfibers, ie 150 ⁇ .
• the drawn pipettes were connected to a syringe containing the culture medium (EGM2 ® Promocell), itself connected to a syringe pump to allow the liquid infusion to a physiological speed 50 ⁇ / ⁇ .
• the infusion rate may vary depending on the internal diameter of the microfiber cell.
• the cellular microfibers are cut into pieces a few centimeters long and are placed in culture medium in petri dish 3cm, under a binocular loupe. They are then connected to the tip of the Pasteur pipettes stretched.
• the complete system (microfibre cell / culture medium, stretched pipette, syringe) is then re-cultured (37 ° C incubator, 5% C0 2) and allows the continuously ® EGM2 medium perfusion in the vascular tubes.
• - Positive control cell-free alginate tube + FITC-Dextran 20kDa: Dextran molecules, of low molecular weight, easily diffuse through the pores of alginate; - Microfiber alginate / HUVEC / SMC according to the invention + FITC Dextran 20kDa: Dextran molecules, low molecular weight, do not diffuse or very little through the cell layers, which make the microfiber waterproof;
• HUVEC / SMC microfibre according to the invention (after hydrolysis of the outer layer of alginate) + FITC Dextran 20kDa: the diffusion rate of the Dextran molecules through the cell layers is close to that observed for the microfiber according to US Pat. the invention further comprising the outer layer of alginate.
• Three hollow cell microfibers were fabricated according to the protocol described in Example 1, varying the extrusion rates of the sorbitol solution and the cell solution for a constant alginate extrusion rate.
• the extrusion rates for the three hollow cell microfibers are summarized in the table below.
• the purpose of this experiment is to verify the reproducibility of the dimensions of the hollow cell microfibers with identical parameters, the impact of the flow rates on the thickness of the external wall of alginate.
• Hollow cellular microfibers were manufactured, according to the protocol described in Example 1, by modifying the outlet tip of the solutions of the concentric coextrusion system (see tip / coextrusion tip 6, FIG. obtain an outlet tip diameter 300 ⁇ , 350 ⁇ , 450 ⁇ and 900 ⁇ . With the 900 ⁇ tip, the alginate solution was extruded alone, to produce empty alginate tubes (without cell suspension).
• the outer diameter and the internal diameter that is to say the light of the microfibres, were measured after synthesis of said microfibers.
• results shown in the table below and in FIG. 5 confirm that it is possible to modify the dimensions of the microfibers and to modify the diameter of the coextrusion spike of the coextrusion system.
• obtaining a hollow alginate tube with a diameter of 900 ⁇ and an outlet tip of 900 ⁇ confirms that the process according to the invention makes it possible to obtain hollow cell microfibers that are perfectly of diameter. perfectly controlled.
• Hollow cellular microfibers with an internal diameter of approximately 400 ⁇ were produced according to Example 1.
• the microfibers are incubated for 45 min in the presence of a calcium-sensitive fluorescent probe, Fluo-4 AM (Thermo Fisher Scientific, F23917, 50 ⁇ g dissolved in 4 ⁇ l of Pluronic acid-at 20% in DMSO-, then diluted in 800 ⁇ l of EGM2, final concentration: 50 ⁇ l), at 37 ° C.
• the AM group acetoxymethyl allows the molecule to cross the plasma membrane, it is cleaved by intracellular esterases which traps the probe in the cytoplasmic compartment.
• the variations of the fluorescence signal intensity provide information on the qualitative variations (non-ratiometric probe) of free calcium accessible at the binding site of the molecule. This information is an indirect measure of activation of signaling pathways involving extracellular calcium entry, and / or release of endoplasmic reticulum calcium stocks.
• the microfibers After rinsing in EGM2 culture medium, the microfibers are imaged epifluorescence with a stereo-microscope.
• a vasoconstrictor specific for the blood vessels, endothelin 1 (ET1, 0.1 ⁇ ) is applied in the vicinity of the tube, in the culture medium.
• the fluorescence signal is collected before, during and after the application of the vasoconstrictor.
• the collected data make it possible to measure: 1 / the contraction of the microfibers (measurement of the external diameter), 2 / the fluorescence signal intensity variations of the Fluo-4 AM, (the intracellular calcium is the second messenger involved in the cascade of signaling triggering the contraction of muscle fibers and thus the decrease of the internal diameter of the vesseloid).
• Endotheline 1 causes the contraction of the microfibers, and a significant decrease in the internal diameter of about 5% ( Figure 7).

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• 2017
  • 2017-03-09 FR FR1751941A patent/FR3063736B1/fr not_active Expired - Fee Related
• 2018
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