Patent Application
20110151564
This invention relates to a method for proliferation of cells on polyelectrolyte multilayer films and use thereof, notably for the preparation of cellular biomaterials.
Polyelectrolytes are polymers whose monomers carry an electrolyte group. These polymers are therefore charged. The layer-by-layer deposition of polyelectrolytes is a simple method for devising surfaces that have special properties [a) G. Decher, J. B. Schlenoff, Multilayer thin films: Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim, 2003. b) G. Decher, Science 277, 1232, 1997]. By successive immersion or deposition of a substrate alternately in a solution of polyanions and of polycations, an assembly is prepared: substrate and multilayer film of polyelectrolytes, in which the anionic and cationic layers alternate. The driving force of the growth of these multilayer films is the excess charges that appear after each new deposition of a polyelectrolyte and thus permit renewed interaction with the polyelectrolyte of opposite sign. This method of treatment is simple to use, is applicable regardless of the geometry of the substrate and generally only employs aqueous solutions. The physicochemical, viscoelastic, structural, surface roughness and wettability properties of the assembly of substrate and polyelectrolyte multilayer film can be adjusted depending on the required use [A. Izquierdo, S. Ono, J. C. Voegel, P. Schaaf, G. Decher, Langmuir, 21, 7558, 2005].
The use of polyelectrolyte multilayer films makes the functionalization of surfaces possible. Improvement of the interaction between cells and surfaces is important in the fields of medicine, biomaterials and biotechnology.
There are electrostatic interactions between negatively charged substrates (for example glass or expanded polytetrafluoroethylene ePTFE) and cells with an overall negative charge, which is unfavourable for adhesion of cells on these substrates. Formation of a polyelectrolyte multilayer film on a biomaterial can promote cellular adhesion and proliferation.
Polyelectrolyte multilayer films have been used for the proliferation of differentiated cells, for example endothelial cells on a glass slide as substrate [C. Boura, P. Menu, E. Payan, C. Picart, J. C. Voegel, S. Muller, J. F. Stoltz, Biomaterials 24, 3521, 2003] and nerve cells on a substrate of TCPS (polystyrene “treated for cell culture”) [S. Forry, D. Reyes, M. Gaitan, L. Locascio, Langmuir 22, 5770, 2006].
Other techniques are used in the prior art for promoting the adhesion of cells on substrates, in particular covering of the substrates with constituents of the extracellular matrix such as: collagen [H. Itoh, Y. Aso, M. Furuse, Y. Noishiki, T. Miyata, Artif. Organs, 25, 213, 2001], fibronectin [A. Rademacher, M. Paulitschke, R. Meyer, R. Hetzer, Int. J. Artif. Organs, 24, 235, 2001], laminin [A. Sank, K. Rostami, F. Weaver, D. Ertl, A. Yellin, M. Nimni, T. L. Tuan. Am. J. Surg. 164, 199, 1992], gelatin [J. S. Budd, P. R. Bell, R. F. James. Br. J. Surg. 76, 1259, 1989], polylysine [a) J. S. Budd, P. R. Bell, R. F. James, Br. J. Surg. 76, 1259, 1989, b), G. Stansby, N. Shukla, B. Fuller, G. Hamilton. Br. J. Surg. 78, 1189, 1991]. Fibronectin is still the most effective protein for enhancing cellular attachment and retention. Works published following clinical studies have shown considerable hydrolysis of fibronectin, which is rather incompatible with use of this protein in vivo [A. Tiwari, H. J. Salacinski, G. Punshon, G. Hamilton, A. M. Seifalian, FASEB J. 16, 791, 2002]. Improvement of the adhesion of cells on substrates for the preparation of grafts for use in vivo is therefore necessary.
Moreover, the use of these various heterologous constituents (of human origin or often of animal origin) and the need for long proliferation times are sometimes incompatible with therapeutic requirements (for example artificial vessels or skin graft).
Furthermore, the techniques of cellular proliferation for the preparation of grafts are carried out in two stages with the techniques known by a person skilled in the art: a first stage of maturation, proliferation, and differentiation of stem cells and/or the expansion of differentiated cells on a first substrate, then detachment of the cells and seeding on another substrate which will be grafted. The need to use two substrates, and therefore to have to detach and then reseed the cells, is time-consuming and increases the risks of contamination.
One aspect of the invention is to provide a method for proliferation of differentiated or undifferentiated cells, which is quick enough for the preparation of grafts.
One aspect of the invention is to provide a method for proliferation of differentiated or undifferentiated cells, in which the proliferation, the maturation and optionally the differentiation of the cells take place on the same substrate as the one that is to be grafted.
Another aspect of the invention is to provide materials covered with viable cells, such as artificial skin or substitutes for vessels or arteries.
In one of these most general aspects, the invention relates to the use of an assembly comprising a substrate and polyelectrolyte multilayer films deposited on said substrate,
In the case of stem cells, the method additionally comprises a stage of differentiation, which also takes place on the aforementioned assembly.
One aspect of the invention relates to the use of polyelectrolyte multilayer films deposited on a substrate, said multilayer films:
The invention is based on the finding that the production of a layer of viable, confluent and adherent cells is quicker than with the techniques known by a person skilled in the art.
In the particular case of application of the invention for the preparation of tissues that will be used as grafts (for example substitutes for vessels or arteries), the invention is based on demonstration of the saving in time and money provided by the use of polyelectrolyte multilayer films for the maturation, the proliferation, and the differentiation of stem cells and/or for the proliferation of differentiated cells. In fact, the maturation, proliferation and differentiation of stem cells and/or the proliferation of differentiated cells can be carried out directly on the substrate that will be used for the graft, in contrast to the techniques known by a person skilled in the art, which are carried out in two stages:
“Substrate” means any material on which the layer-by-layer deposition of polyelectrolytes can be carried out.
“Polyelectrolytes” means polymers whose monomers carry an electrolyte group. “Polyelectrolyte multilayer films” means the stack of layers obtained by the layer-by-layer deposition of polyelectrolytes [G. Decher, J. B. Schlenoff, Multilayer thin films: Sequential Assembly of Nanocomposite Materials, Wiley-VCH, Weinheim, 2003].
“Top layer of polyelectrolytes” means the last layer of polyelectrolytes deposited by the technique of layer-by-layer deposition.
“Inner layers of polyelectrolytes” means the layers of polyelectrolytes located between the substrate and the top layer of polyelectrolytes.
“Polycation” means a polymer with an overall positive charge, “with an overall positive charge” meaning that the total charge is positive, but this does not exclude the presence of negatively charged monomers in the polymer.
“Polyanion” means a polymer with an overall negative charge, “with an overall negative charge” meaning that the total charge is negative, but this does not exclude the presence of positively charged monomers in the polymer.
“Biological molecules” means molecules that participate in the metabolic process and in the maintenance of a living organism, for example proteins, DNA, RNA, cytokines, growth factors, for example those necessary for the recruitment and the differentiation of the desired cell type (notably VEGF in the case of the vascular cells).
“Biologically active molecules” means molecules that have curative or preventive properties, for example which accelerate or reduce cell differentiation and/or proliferation, or for example medicinal products (notably VEGF in the case of ischaemia, or taxol in the case of cancers).
The expression “multilayer films coated with an assembly of molecules” denotes that an assembly of molecules is deposited on the surface of the multilayer films. The molecules can be adsorbed on the surface. Interactions occur between the molecules and the top layer of polyelectrolytes, but the molecules can also be buried between the inner polyelectrolyte layers of the multilayer film. For example, in the case when the molecule is a protein with an overall positive charge coating a polyelectrolyte multilayer film whose top layer of polyelectrolytes is a polyanion, the principal electrostatic interactions will be those between the positive charges of the protein and the negative charges of the top layer of polyelectrolytes. However, a protein with an overall positive charge can contain negatively charged amino acids, which do not interact with the top layer of the polyelectrolyte, but instead with the positively charged polyelectrolytes of the inner layer of polyelectrolytes.
The expression “completely coated” means that the molecules coat the entire surface of the polyelectrolyte multilayer film. The expression “partially coated” means that the molecules are only present at certain places on the polyelectrolyte multilayer film. This partial coating can be obtained by spraying techniques, such as those used in the publications [Porcel et al., Langmuir 22, 4376-83, 2006 and Porcel et al., Langmuir 21, 800-02, 2005]. Images obtained with the laser fluorescence microscope or atomic force microscope can make it possible to determine whether the coating is partial or complete.
The expression “comprising biological or biologically active molecules” means that molecules are present in the polyelectrolyte multilayer film. These molecules are incorporated between the layers of polyelectrolytes of the polyelectrolyte multilayer film. The techniques for incorporating molecules between polyelectrolytes are explained in the publications of N. Jessel, M. Oulad-Abdelghani, F. Meyer, P. Lavalle, Y. Haîkel, P. Schaaf, J. C. Voegel, PNAS 103, 8618, 2006 (example of incorporation of a biologically active molecule, β-cyclodextrin) and of A. Dierich, E. Le Guen, N. Messaddeq, J. F. Stoltz, P. Netter, P. Schaaf, J. C. Voegel, N. Benkirane-Jessel, Adv. Mater. 16, 693, 2007 (example of incorporation of growth factors TGFβ1).
“Adjacent layers” means two layers of polyelectrolytes that were deposited one after another during formation of the polyelectrolyte multilayer film.
“Properties of the polyelectrolyte multilayer film” means the physicochemical properties, notably the viscoelasticity, surface roughness and wettability of the polyelectrolyte multilayer film.
“Biological properties of said molecules” means the curative or preventive properties of the biologically active molecules.
“Proliferation of cells” means the division and maturation of cells.
“Initial cells” means the cells that are brought in contact initially with the polyelectrolyte multilayer film.
“Covering of the multilayer films with cells” means the production of a layer, preferably a monolayer, of cells, deposited on the polyelectrolyte multilayer film. The cells can be adsorbed on the top layer of polyelectrolytes of the multilayer film, but there may also be interactions with inner layers of polyelectrolytes of the polyelectrolyte multilayer film. These interactions can for example be ionic bonds, hydrogen bonds, van der Waals bonds etc.
“Adherent cells” means cells that adhere to the polyelectrolyte multilayer film or to any biological or biologically active molecules with which it is coated. This adhesion can for example be visualized by images of histological sections or from observation with the scanning electron microscope and can be confirmed via the expression of specific markers of the cells (for example, integrins and the arrangement of the cytoskeleton).
“Viable cells” means cells that are capable of surviving. Cell viability can for example be determined by the ABRA test (Alamar Blue® redox assay).
“Confluent cells” means cells whose cell membranes are in contact. This occurs when the initial cells put in culture have proliferated so as to occupy all the available space in a monolayer. Confluence can be detected from images obtained in phase-contrast or laser-scanning microscopy.
“Cells resulting from proliferation of the initial cells” means the cells resulting from the division, maturation, and optionally differentiation (when the initial cells are stem cells) of the initial cells.
Another aspect of the invention relates to the use of polyelectrolyte multilayer films deposited on a substrate, said multilayer films:
The expression “chemical bond not being of a covalent nature” means that the bonds between the molecules and the layers of polyelectrolytes are, for example, ionic bonds, hydrogen bonds, or van der Waals bonds, which do not alter the properties of the molecules and of the polyelectrolyte multilayer film.
According to one aspect of the invention, the top layer of polyelectrolytes of the multilayer films is a polycation and the multilayer films are not coated with a collection of biological or biologically active molecules, and do not contain biological or biologically active molecules incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films.
When the top layer of polyelectrolytes is positively charged, the cells, whose membrane is negatively charged, generally adhere to the polyelectrolyte multilayer film.
According to another aspect of the invention, the top layer of polyelectrolytes of the multilayer films is a polyanion and the multilayer films are not coated with a collection of biological or biologically active molecules, and do not contain biological or biologically active molecules incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films.
When the top layer of polyelectrolytes is negatively charged, the cells, whose membrane is negatively charged, generally do not adhere to the polyelectrolyte multilayer film (repulsive electrostatic interactions).
These last two cases correspond to the use of a polyelectrolyte multilayer film for cellular proliferation without intervention of biological or biologically active molecules.
According to one aspect of the invention, the top layer of polyelectrolytes of the multilayer films is a polycation and the multilayer films are coated with a collection of biological or biologically active molecules and optionally contain biological or biologically active molecules incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films.
According to another aspect of the invention, the top layer of polyelectrolytes of the multilayer films is a polyanion and the multilayer films are coated with a collection of biological or biologically active molecules and optionally contain biological or biologically active molecules incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films.
These last two aspects relate to different cases:
When the top layer of polyelectrolytes is negatively charged, and therefore when the cells do not adhere to the multilayer film, coating of the polyelectrolyte multilayer film with biological molecules is particularly advantageous as it can make it possible to reverse the polarity of the substrate and therefore promote adhesion of the cells.
For example, the top layer of a (PAH-PSS)3 multilayer film is negatively charged and the cells do not generally adhere. If the multilayer film is covered with proteins with an overall positive charge, the polarity of the surface is reversed and adhesion of the cells is promoted.
In the present invention and according to an advantageous embodiment, the initial cells are differentiated cells, notably selected from keratinocytes, chondrocytes, nerve cells, dendritic cells, endothelial cells, fibroblasts, epiblasts, myoblasts, cardiomyoblasts, myocytes, epithelial cells, osteocytes, osteoblasts, hepatocytes and cells of the islets of Langerhans.
According to another embodiment of the invention, the initial cells are stem cells, notably selected from totipotent, pluripotent and multipotent cells.
“Totipotent cells” means cells that can be differentiated into any cell type of the organism. They permit the development of a complete individual.
“Pluripotent cells” means cells that can be differentiated into cells derived from any of the three germ layers. They cannot produce a complete organism.
“Multipotent cells” means cells that can be differentiated into several types of differentiated cells but only for particular types of cells. For example, haematopoietic multipotent cells can differentiate into red blood cells, platelets, lymphocytes or macrophages but they cannot differentiate into muscle cells.
As examples of stem cells, we may mention embryonic and haematopoietic stem cells, mesenchymal cells, precursors such as EPCs (endothelial progenitor cells).
According to another advantageous embodiment of the invention, the polyelectrolyte multilayer films are constituted of alternating layers of polycations and polyanions,
According to another advantageous embodiment of the invention, the number of layers of the polyelectrolyte multilayer films is from 3 to 100, in particular 3 to 50, notably 5 to 10 and in particular 7.
Below 7 layers, the film is still permeable to small molecules, for example to Hoechst 33258 (molecular weight 623 Da).
According to another advantageous embodiment of the invention, the polyelectrolyte multilayer films are selected from (PAH-PSS)3, (PAH-PSS)3-PAH and PEI-(PSS-PAH)3. [a) H. Kerdjoudj et al. Bio-Medical Materials and Engineering, 16(4), 123, 2006 b) C. Boura et al. Biomaterials 26, 4568, 2005].
According to another advantageous embodiment of the invention, the substrate is a synthetic substrate advantageously selected from glass, TCPS (“treated cell culture” polystyrene), polysiloxane, perfluoroalkyl polyethers, biocompatible polymers especially Dacron®, polyurethane, polydimethylsiloxane, polyvinyl chloride, Silastic®, polytetrafluoroethylene (ePTFE), and any material used for prostheses and/or implanted systems.
According to another advantageous embodiment of the invention, the substrate is a natural substrate advantageously selected from blood vessels, veins, arteries, notably decellularized, notably de-endothelialized umbilical arteries, said vessels, veins and arteries being obtained from organs from donors or from animals.
According to another advantageous embodiment of the invention, the substrate is a natural substrate advantageously selected from the placental dermis, the bladder or any other substrate (organ) of human or animal origin.
According to an advantageous embodiment of the present invention, the polyelectrolyte multilayer films deposited on a substrate are sufficiently rigid to permit the adhesion of cells and sufficiently flexible to deform under the action of arterial pulsations and withstand physiological pressures from 10 to 300 mmHg, notably 50 to 250 mmHg and advantageously 80 to 230 mmHg.
This pressure range corresponds to that observed for physiological pressures. In humans, hypertension is said to be severe if the systolic pressure is above 180 mmHg Hypotension refers to systolic pressure below 50 mmHg.
“Physiological pressures” means the pressures of the blood in the arteries, veins and vessels in a healthy subject.
According to an advantageous embodiment of the present invention, the covering of the polyelectrolyte multilayer films deposited on the substrate with the adherent cells is such that it withstands the shearing action of the blood flow, notably in vivo.
“Shearing action of the blood flow” means the frictional tangential force induced by the blood flow that is exerted on the polyelectrolyte multilayer film when the assembly: substrate, polyelectrolyte multilayer film, and cells covering it, is in physiological conditions.
According to another advantageous embodiment, the invention makes it possible to prepare vascular endoprostheses, balloons for angioplasty, artificial arteries or vessels for grafts, vascular shunts, heart valves, artificial components for the heart, pacemakers, ventricular assist devices, catheters, contact lenses, intraocular lenses, matrices for tissue engineering, biomedical membranes, dialysis membranes, membranes for cell encapsulation, prostheses for cosmetic surgery, orthopaedic prostheses, dental prostheses, dressings, sutures, diagnostic biosensors.
The invention also relates to a method of covering initial cells, stem cells or differentiated cells, comprising:
At the end of the process, the cells may or may not be detached from the polyelectrolyte multilayer film. For example, for the preparation of artificial skin, the cells will be detached from the multilayer film. Conversely, for the preparation of vascular or arterial substitutes, the endothelial cells are not detached, provided that the substrate is biocompatible, since the assembly: biocompatible substrate/polyelectrolyte multilayer film/endothelial cells, is grafted.
“Biocompatible substrate” means a substrate that is well tolerated by a living organism, which does not cause rejection, toxic reactions, lesions or a harmful effect on the biological functions of the organism.
According to an advantageous embodiment of the present invention, the method is a method of covering initial stem cells comprising:
In this case the initial cells are stem cells. It was found, unexpectedly, that the stem cells can proliferate and differentiate up to confluence in a shorter time than in the methods of the prior art. The time taken in the invention is 14 days, notably 11 days, in particular 7 days. For example, on a glass slide substrate and with the (PAH-PSS)3-PAH polyelectrolyte multilayer film, confluence is reached in 14 days whereas it takes 60 days when using fibronectin (which is the protein giving the quickest proliferation and differentiation times among the techniques known by a person skilled in the art).
According to an advantageous embodiment of the present invention, the method is a method of covering differentiated initial cells comprising:
In this case the initial cells are differentiated cells. It was found, unexpectedly, that the initial cells can proliferate up to confluence in a shorter time than in the methods of the prior art. The time taken in the invention is 7 days, notably 5 days, in particular 3 days. For example, on the ePTFE substrate and with the PEI-(PSS-PAH)3 polyelectrolyte multilayer film, confluence is reached in 7 days or less, whereas without deposition of a polyelectrolyte multilayer film, no cells adhere.
According to an advantageous embodiment, in the method of the invention the multilayer films are coated with a collection of biological or biologically active molecules, and/or contain biological or biologically active molecules, incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films, the incorporation being such that neither the properties of the polyelectrolyte multilayer film, nor the possible biological properties of said molecules are altered.
According to an advantageous embodiment of the present invention, the method comprises:
In this case, the adherent, viable and confluent cells are detached from the polyelectrolyte multilayer film.
According to an advantageous embodiment of the present invention, the method comprises:
In this case, the adherent, viable and confluent cells are not detached from the polyelectrolyte multilayer film.
According to a preferred embodiment, the method is a method of covering endothelial initial cells which comprises:
This case corresponds to a method for proliferation of endothelial cells on a polyelectrolyte multilayer film for the preparation of vascular or arterial substitutes which will be used as grafts. The use of polyelectrolyte multilayer films offers many advantages. Thus, the assembly of artery or vessel substrate/polyelectrolyte multilayer film is sufficiently rigid to permit adhesion of the cells and sufficiently elastic to withstand the deformation caused by the blood flow. Moreover, the monolayer of cells obtained must allow the passage of oxygen and nutrients, which should permit the essential exchanges between the blood and the surrounding tissues.
According to another preferred embodiment, the method is a method of covering initial stem cells comprising:
This case corresponds to a method of proliferation of stem cells, then differentiation into endothelial cells, on a polyelectrolyte multilayer film for the preparation of vascular or arterial substitutes which will be used as grafts.
In the method of the invention, the initial stem cells are notably selected from totipotent, pluripotent and multipotent cells.
In the method of the invention, the differentiated initial cells are notably selected from keratinocytes, chondrocytes, nerve cells, dendritic cells, endothelial cells, fibroblasts, epiblasts, myoblasts, cardiomyoblasts, myocytes, epithelial cells, osteocytes, osteoblasts, hepatocytes and cells of the islets of Langerhans.
According to a particular embodiment, in the method of the invention, the polyelectrolyte multilayer films are constituted of layers, preferably alternating, of polycations and of polyanions,
Advantageously, the number of layers of said polyelectrolyte multilayer films is from 3 to 100, in particular 3 to 50, notably 5 to 10 and in particular 7.
According to another embodiment of the invention, in the method of the invention the polyelectrolyte multilayer films are selected from (PAH-PSS)3, (PAH-PSS)3-PAH and PEI-(PSS-PAH)3.
According to another embodiment of the invention, in the method of the invention the substrate is selected from synthetic substrates such as glass, TCPS (“treated cell culture” polystyrene), polysiloxane, perfluoroalkyl polyethers, biocompatible polymers especially Dacron®, polyurethane, polydimethylsiloxane, polyvinyl chloride, Silastic®, polytetrafluoroethylene (ePTFE) and any material used for prostheses and/or implanted systems.
According to another embodiment of the invention, in the method of the invention the substrate is selected from natural substrates such as blood vessels, veins, arteries, notably decellularized, notably de-endothelialized umbilical arteries, said vessels, veins and arteries being obtained from organs of donors or of animals.
According to another advantageous embodiment of the invention, the substrate is a natural substrate advantageously selected from the placental dermis, the bladder or any other substrate (organ) of human or animal origin.
The present invention relates to a composition comprising:
According to another embodiment, the composition of the invention comprises a substrate, polyelectrolyte multilayer films deposited on said substrate, said multilayer films are coated with a collection of biological or biologically active molecules and/or contain biological or biologically active molecules, incorporated between at least two adjacent layers of the aforesaid polyelectrolyte multilayer films.
According to another embodiment, the composition of the invention comprises a substrate, polyelectrolyte multilayer films deposited on said substrate, and a layer of stem cells covering said polyelectrolyte multilayer film.
According to a particular embodiment, the initial stem cells are notably selected from totipotent, pluripotent and multipotent cells.
According to another embodiment, the composition of the invention comprises:
According to an advantageous embodiment, the composition of the invention comprises:
According to another advantageous embodiment, the composition of the invention comprises:
According to another embodiment, the composition of the invention comprises:
In the composition of the invention defined above, the substrate is a synthetic or natural substrate, and in particular a synthetic substrate.
In the compositions of the invention, the differentiated initial cells are notably selected from keratinocytes, chondrocytes, nerve cells, dendritic cells, endothelial cells, fibroblasts, epiblasts, myoblasts, cardiomyoblasts, myocytes, epithelial cells, osteocytes, osteoblasts, hepatocytes and cells of the islets of Langerhans.
In the compositions of the invention, the polyelectrolyte multilayer films are constituted of layers, preferably alternating, of polycations and of polyanions,
According to an advantageous embodiment, in the compositions of the invention the number of layers of said polyelectrolyte multilayer films is from 3 to 100, in particular 3 to 50, notably 5 to 10 and in particular 7.
According to an advantageous embodiment, in the compositions of the invention the polyelectrolyte multilayer films are selected from (PAH-PSS)3, (PAH-PSS)3-PAH and PEI-(PSS-PAH)3.
In the compositions of the invention, the substrate is selected from natural substrates, such as blood vessels, veins, arteries, notably decellularized, notably de-endothelialized umbilical arteries, said vessels, veins and arteries being obtained from organs of donors or of animals, and the placental dermis. (idem)
In the compositions of the invention, the substrate is advantageously selected from the placental dermis, the bladder or any other substrate (organ) of human or animal origin.
In the compositions of the invention, the substrate is selected from synthetic substrates, notably glass, TCPS (“treated cell culture” polystyrene), polysiloxane, perfluoroalkyl polyethers, biocompatible polymers especially Dacron®, polyurethane, polydimethylsiloxane, polyvinyl chloride, Silastic®, polytetrafluoroethylene (ePTFE) and any material used for prostheses and/or implanted systems.
For
In
In
The glass slides are washed to reveal the silica (Si-) and to make the surface of the slides negative.
More precisely, the glass slides are washed for 15 min at 100° C. in a 0.01 M solution of sodium dodecyl sulphate (SDS). Three washings are then carried out with filtered distilled water. The slides are then immersed in 0.12 M hydrochloric acid solution for 15 min at 100° C. Three washings are carried out with filtered distilled water. The slides are stored at 4° C. in filtered distilled water before treatment.
1.1.2. Preparation of the ePTFE
Patches of expanded polytetrafluoroethylene ePTFE with diameter of 9 mm are prepared from tubular vascular prostheses of ePTFE (6 mm inside diameter and fibril length 25 μm). These patches are then glued in 48-well culture plates. The polyelectrolyte multilayer films are then constructed directly on the ePTFE inside the wells. Preliminary studies showed absence of cytotoxicity of the glue.
When RPMI 1640/M199 mixture is supplemented with these additives, it forms the so-called “complete” medium. The shelf life of the complete medium, stored at 4° C., does not exceed 2 weeks.
The arteries are recovered from the human umbilical cord. Using two surgical forceps, the umbilical cord is dilacerated and lengths of arteries of at least 6 cm are isolated and immersed in buffer (Hank's Balanced Salt Solution HBSS). After rinsing several times, generally three to five (until the artery no longer contains blood) the arteries are put in cryotubes containing 1 mL of a freezing solution, which is constituted of 70% of complete medium supplemented with 10% of dimethylsulphoxide (DMSO, Sigma, France) and 20% of fetal calf serum (Gibco BRL, France), previously decomplemented at 56° C. for 30 min.
The cryotubes are stored overnight at −80° C., and then immersed in liquid nitrogen at −180° C. The shelf life is normally 6 months (the time required for carrying out serological tests when using allografts taken from cadavers).
The umbilical arteries are thawed by immersing the cryotubes in a water bath at 37° C. They are then washed with a decontaminating solution, which is constituted of RPMI 1640 medium (Gibco BRL, France) supplemented with 100 IU/mL of penicillin (Gibco BRL, France), 100 μg/mL of streptomycin (Gibco BRL, France) and 2.5 μg/mL of Fungizone® (Gibco BRL, France).
The lumen of the artery is washed three times with buffer (HBSS), and then it is filled with a digesting solution (trypsin/EDTA 0.25%). After incubation at 37° C. for 20 min, the artery is washed with 2 mL of medium containing whole serum. The arteries called “de-endothelialized arteries” hereinafter are those that have undergone this process of cryopreservation.
The polyelectrolyte multilayer films are constituted of alternating solutions of polycations and polyanions.
The use of these polyelectrolytes is described in the following works:
The polyelectrolyte multilayer films were deposited in the lumen of the previously de-endothelialized umbilical arteries, on glass slides or on ePTFE, as appropriate. Assembly is carried out at room temperature by successive depositions of the substrate alternately in a solution of polycation and of polyanion. After washing twice with Tris/NaCl buffer for the glass and arteries as substrates, and with distilled water for the ePTFE substrate, the substrates are brought in contact with
Before each experiment, the cell culture plates containing the glass slides are exposed to UV for 15 min for sterilization.
Glass slides covered with fibronectin (Sigma, France) at a concentration of less than 250 μg/mL are used as positive controls.
* In the Case of ePTFE on Which PEI-(PSS-PAH)3 Has Been Deposited
The ePTFE substrate on which a PEI-(PSS-PAH)3 polyelectrolyte multilayer film has been deposited is dried at least overnight at 4° C. after deposition of the multilayer film and prior to use. It is stored for at most 15 days at 4° C.
The TCPS substrate (Tissue Culture Polystyrene Surface) is the material most commonly used for cell culture, and it is a polymer that is widely used for studying the mechanisms of interactions between cells and artificial material. It is used as a positive control.
The de-endothelialized arteries on which no polyelectrolyte multilayer film had been deposited, are submitted to several injections of washing buffer and are regarded as controls (control artery). The arteries on which polyelectrolyte multilayer films had been deposited and the control arteries are stored overnight at 4° C. in a decontaminating solution before use. The latter is constituted of RPMI 1640 medium (Gibco BRL, France) supplemented with 100 IU/mL of penicillin (Gibco BRL, France), 100 μg/mL of streptomycin (Gibco BRL, France) and 2.5 μg/mL of Fungizone® (Gibco BRL, France).
1.3.1. When the Substrate is ePTFE
Verification of uniform deposition of the polyelectrolyte multilayer film on the ePTFE substrate was carried out by confocal laser scanning microscopy.
Verification of uniform deposition of the polyelectrolyte multilayer film on the internal surface of vessels was carried out by confocal laser scanning microscopy.
The mechanical tests are carried out by means of a test bench developed in the laboratory. The pressure is supplied by a pressure detector (XTC-190M-0.35 BARVG, Kulite, Inc) located at pump outlet (EX303C-50, Prodera, France). The information is representative of the pressure exerted on the inside walls of the artery. The outside diameter of the artery is evaluated by a CCD camera (FZS 1024, Sensopart UK Ltd), which measures its deformation. The CCD unit delivers a voltage in relation to the amount of light received by a neon lamp, which serves as the standard light source.
Each end of the artery (treated and control) is mounted in plastic tips, then the artery is fixed in a plexiglas chamber filled with physiological saline solution preheated to 37° C. The artery must be kept taut. Using a syringe fitted with a tube, the interior of the artery is filled with physiological saline solution, avoiding the formation of air bubbles. The free end of the artery is clamped to close the circuit. The pump thus increases the pressure in this closed circuit.
The parameters are entered in software for controlling the pump. The pressure in the artery increases by constant steps every 15 seconds up to 230 mmHg (with increments of 30 mmHg). The outside diameter of the artery is recorded for each pressure.
The percentage deformation is calculated according to the following equation:
ΔD=100×(Dp−DO)/DO
The elasticity of the arteries corresponds to the straight line ΔD over pressure, measured at physiological pressures (between 80 and 150 mmHg).
The endothelial cells required for this study are obtained from human umbilical veins (HUVECs Human Umbilical Vein Endothelial Cells). They are taken from umbilical cords of neonates (donated by the Nancy District Maternity Hospital). The cords are obtained from healthy donors, after their consent. Collected immediately after delivery of the placenta, the cord is cut to a size of 20 to 25 cm and immediately put in a 75 cm2 culture bottle containing 150 mL of sterile HBSS. Quickly cooled to 4° C., the cord is used as soon as possible. It can be kept for 4-6 hours.
The cells are cultured according to Jaffe's method (E. A. Jaffe, R. L. Nachman, C. G. Becker, C. R. Minick, J Clin Invest. “Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.” 52(11), 2745-56, 1973) in several stages:
The HBSS buffer is removed from the flask and the cord is placed in a sterile culture flask. The exterior of the cord is cleaned with 75% ethanol. A tap is fixed to one of the orifices of the umbilical vein and tied firmly to the cord. Using a syringe, the vein is washed three times with HBSS buffer (filtered and preheated to 37° C.) to remove the blood from it. Then the other end of the cord is clamped.
15-20 mL of the digesting solution (filtered and preheated to 37° C.) is injected into the vein until it is sufficiently dilated. The cord is immersed in 200 mL of HBSS; the whole is put in a water bath at 37° C. for 10 minutes. The cord is then gently placed in a culture flask and massaged for a few seconds. It is then unclamped above a 50 mL plastic tube (Polylabo, France) containing 20 mL of complete medium to stop the action of the trypsin, and in which the digesting solution containing the free endothelial cells is collected. The vein is then rinsed with HBSS buffer so that any cells still present are entrained into the tube. The cellular suspension is centrifuged at 1200 rpm (300 g) for 6 minutes, at room temperature. After centrifugation and deposition of the supernatant, the cellular pellet is resuspended in 10 mL of HBSS. Then a second centrifugation is carried out. The cells are resuspended in 5 mL of complete medium, sown in a 25 cm2 culture bottle and put in an incubator at 37° C. (5% CO2 and 95% air) and at saturation humidity.
The day following extraction of the endothelial cells, the cells are washed twice with HBSS buffer, with small oscillating movements so as to remove the red blood cells. Then the cells are put back in the incubator with 5 mL of complete medium. The medium is renewed every other day. Normally, the cells are confluent after 5-7 days.
At confluence, the cells are washed twice with 5 mL of HBSS (preheated to 37° C.) and put in contact with 5 mL of trypsin-EDTA 0.125% (filtered), at 37° C., for 3 minutes.
The digesting action of the trypsin is stopped by adding 5 mL of complete medium. The cellular suspension is collected in sterile conical tubes and then centrifuged at 1200 rpm (300 g) for 6 minutes. The cells are then resuspended in 5 mL of complete medium.
3.1.5. Seeding of the Endothelial Cells on ePTFE on Which a Polyelectrolyte Multilayer Film Has Been Deposited
The cells are sown, after their second passage (P2), at a cell density of 5.104 cells/well on ePTFE on which the PEI-(PSS-PAH)3 polyelectrolyte multilayer film was deposited, on ePTFE on which a PAH monolayer was deposited, on ePTFE alone and on TCPS (Tissue Culture Polystyrene Surface) (positive control). The medium is changed every 3 days.
3.2.1 Evaluation of Cell Viability with Alamar blue®
Seifalian et al. (A. M. Seifalian, H. J. Salacinski, G. Punshon, B. Krijgsman, G. Hamilton, J. Biomed. Mater. Res. “A new technique for measuring the cell growth and metabolism of endothelial cells seeded on vascular prostheses.” 15, 55(4), 637-44, 2001) demonstrated that Alamar blue (Serotec Ltd, Kidlington, England) is an agent that has many advantages in evaluation of the metabolism of endothelial cells and therefore of the viability of the cells growing on vascular prostheses.
Alamar blue® redox assay (ABRA) (Alamar blue test) is a technique that has been used for monitoring the viability of endothelial cells seeded on vascular substitutes (ePTFE). With this technique, cellular proliferation, cytotoxicity and viability can be measured quantitatively. Alamar blue is composed of a redox indicator (colorimetric indicator), which changes colour in relation to chemical reduction of the culture medium. Alamar blue is reduced by mitochondrial activity, which is representative of cellular metabolic activity and therefore of cell viability.
Alamar Blue has interesting properties as it is soluble in the medium, stable in solution, nontoxic to the cells and produces changes that can be measured easily. The test does not require lysis of the cells, which makes it possible to follow the kinetics of the signal.
Measurement of cell viability is therefore based on the degree of oxidoreduction of Alamar blue determined by the difference between densitometric measurement at 570 nm (absorbance of the reduced compound) and at 630 nm (absorbance of the oxidized compound). Taking into account the partial overlap of the absorption spectra of the reduced compound (red) and of the oxidized compound (blue), the absorbance is measured at two wavelengths and the difference in optical density (OD) is determined according to the formula:
ΔOD=[OD(570nm)exp.−OD(630nm)exp.]−[OD(570nm)cont.−OD(630nm)cont.]
exp.=experimental; cont.=control without cells; Δ=difference
The procedure is as follows. The Alamar blue test is carried out according to the chosen protocol. The endothelial cells are sown on the surfaces for 1, 3, or 7 days. At the chosen time, the culture medium is replaced with fresh medium without serum containing 10% v/v of Alamar blue (the sensitivity of the Alamar blue technique depends on the volume ratio between Alamar blue and the DMEM medium (Dulbecco's Modified Eagle Medium) without phenol red (GibcoBRL, France)). 500 μL of this mixture is put in each well. The culture plate is put in the incubator at 37° C.
Densitometric measurement is carried out 3 hours after adding the marker. The difference in optical density (indicator of cell viability) is then determined Wells without cells are used as reference.
After culture for one day, no difference in metabolic activity can be detected.
After culture for 3 days, a significant increase in metabolic activity of the cells is measured on TCPS. On the ePTFE substrates, the metabolic activity remains similar to that observed after culture for one day.
After culture for 7 days, the values of metabolic activity observed for the ePTFE on which the PEI-(PSS-PAH)3 polyelectrolyte multilayer film was deposited (0.59±0.20) are similar to those observed for the TCPS substrate. However, for the same culture time, the values of metabolic activity observed for the ePTFE on which the PAH polyelectrolyte was deposited and for the ePTFE alone are significantly less than those observed for the ePTFE on which the PEI-(PSS-PAH)3 polyelectrolyte multilayer film was deposited.
The endothelial cells therefore began to proliferate on the ePTFE on which the PEI-(PSS-PAH)3 polyelectrolyte multilayer film was deposited after culture for three days and maturation to obtain confluent cells occurs in seven days of culture. Moreover, a low cell density (5.104 cells/cm2) was sufficient to obtain a monolayer of confluent cells.
In contrast, the deposition of a single layer of PAH polyelectrolyte on ePTFE is not sufficient for observing similar cell viability after an identical culture time.
For observation with the electron microscope (STEREOSCAN S 240, CAMBRIDGE (UK)), the cells must be fixed. After washing twice with PBS buffer heated to 37° C., the cells are fixed with 2.5% glutaraldehyde, and stored at 4° C. before observation with the SEM. The samples are then prepared to permit observation in electron microscopy (dehydration, fixation and covering with a layer of gold-palladium). This investigation was carried out in the Electron Microscopy Laboratory (Pr Folliguet, Medical Faculty, Nancy).
The phenotype of the endothelial cells is evaluated by expression of the von Willebrand factor (vWf) in confocal microscopy. For visualization of each cell, the nuclei are labelled with propidium iodide. After 7 days of culture, the endothelial cells are washed with DMEM (Dulbecco's Modified Eagle Medium) without phenol red (Gibco BRL, France) at 37°. They are then fixed immediately with PAF (paraformaldehyde) 1% v/v in PBS. After 10 minutes at room temperature, the PAF is removed and the cells are permeabilized using Triton-X100 (Sigma, France) at 0.5% in PBS. The cells are then incubated for 45 minutes with a mouse vWf anti-human monoclonal antibody (clone F8/86, Dako, Trappes, France) diluted 1/50 in Triton 0.1%. The cells are then washed with DMEM to remove the excess antibodies and are incubated for 30 minutes with a IgG anti-mouse polyclonal antibody conjugated with Alexa Fluor 488 (Molecular Probes, Oregon, USA) diluted 1/100 in DMEM. The isotypic control is prepared in the same conditions. The cells are incubated for 30 minutes with propidium iodide (PI) (λexcitation=535 nm, λemission=617 nm, Molecular Probes, 1 mg/mL in distilled water) diluted 1/1000 in DMEM. The labelled cells are then visualized in the confocal laser scanning microscope using an objective 40 and an He—Ne laser for the 543 nm excitation (PI) and an Ar laser for the 488 nm excitation (vWf).
The cells used are those described in paragraph 3.1.2. “cells used”
An umbilical cord is put in a sterile Petri dish. The exterior of the cord is cleaned with 70° ethanol. The orifice of the umbilical vein is located with forceps in order to insert a sterile tap. To remove the blood from the umbilical vein, the latter is washed three times with HBSS buffer (Hank's Balanced Salt Solution). The umbilical vein is then filled with 15 to 20 mL of a digesting solution preheated to 37° C. (trypsin/EDTA 0.25%). The cord, immersed in HBSS buffer, is put on a water bath. After incubation for 12 min, the digesting solution is collected in a 50 mL bottle containing 5 mL of complete medium. The vein is washed with HBSS buffer. The cellular suspension is centrifuged at 300 g for 10 min at room temperature. The cellular pellet is resuspended in 10 mL of HBSS buffer. After the second washing, the cells are resuspended in 5 mL of complete medium. The endothelial cells (HUVEC) are sown in a 25 cm2 culture bottle, and then are put in an incubator at 37° C. (5% CO2 and 95% air).
When the endothelial cells reach confluence, the culture medium is removed and the cells are washed twice with 5 mL of HBSS buffer without Ca2+ or Mg2+. This washing makes it possible on the one hand to remove the serum, which inhibits the enzymatic activity of the trypsin, and on the other hand to release Ca2+ ions, which in their turn facilitate detachment of the cells. The cells are then detached using 5 mL of solution of Trypsin-EDTA 0.125%. The action of the trypsin is stopped after 2 min at 37° C. by adding 10 mL of complete medium. The cellular suspension is collected in a sterile 50-mL Falcon tube, then centrifuged at 300 g. The cellular pellet is resuspended in complete medium.
At the second passage (P1), the cells are then sown at a cell density of 105 cells/cm2 in the various matrices (arteries on which a (PAH-PSS)3-PAH polyelectrolyte multilayer film was deposited and control arteries). For better distribution of the cells, the endothelialized substitutes are put in sterile Falcon tubes, stirring gently for 4 hours. They are cultured in an incubator at 37° C., 5% CO2 for 7 days.
The retention of the HUVECs sown in the lumen of the arteries is evaluated in a flow chamber developed in the laboratory. The endothelial cells are exposed to laminar flows of 1 Pa (10 dynes/cm2), for one hour.
A peristaltic pump (Ismatech, Switzerland) provides circulation of the culture medium. Upstream of the chamber, two syringes are added to the circuit, for creating a sinusoidal modulation in order to dampen the parasitic fluctuations of the flow. A gas mixture (5% CO2 and 95% air) is introduced into the reservoir of the medium to control the variations in pH. The system is put in a stove set to 37° C.
The shear stress is calculated from the following equation:
τ=4Qμ/πr3
Consequently, knowing the value of the flow rate of the peristaltic pump, it is possible to find the shear stress exerted in the lumen of the artery. The peristaltic pump was calibrated by measuring the flow rate as a function of the rotary speed.
Following this calibration, the following relation was obtained by linear regression:
The results in
In this example, vascular substitutes (umbilical arteries) treated with a (PAH-PSS)3 polyelectrolyte multilayer film are evaluated in an animal (the rabbit). The untreated de-endothelialized arteries are used as control.
All the experiments carried out on the rabbit were conducted observing the current European rules on bioethics (Decree No. 2001-131 of 6 February 2001, linked to European Directive 86-609-EEC of 1986). Thus, the animals (male New Zealand white rabbits, weighing 3±0.25 kg) are of controlled origin (CEGAV, St Mars d'Egrenne, France), and were kept in an approved animal house and all necessary precautions were taken to avoid any suffering of the animal during the experiments.
Induction of anaesthesia is performed via the external marginal vein of the ear, by means of an intravenous catheter (Salva epicranial set, COOPER, Rhône-Poulenc Rorer, Melun, France), by slow injection of a dose of 40 mg/kg of pentobarbital sodium (Ceva Santé Animale, France), diluted to a quarter in physiological serum (NaCl 0.9% Cooper, Rhône-Poulenc, France). In contrast to the volatile anaesthetics, pentobarbital sodium does not seem to alter the behaviour of the polynuclear neutrophils nor of the platelets. The efficacy of anaesthesia is verified before commencement of any surgery by interdigital pinching of the rabbit's hindpaw. Anaesthesia is maintained by intravenous injection (marginal vein of the ear) of pentobarbital diluted to ¼ in physiological serum repeatedly.
The anaesthetized animal is placed in dorsal recumbency on the heated table and its body temperature is maintained at a constant 37° C. The areas for surgical intervention are shaved and then disinfected with iodinated polyvidone (Bétadine dermique 10% ™ Laboratoire Sarget, Mérignac, France).
After local anaesthesia with Xylocaine, an incision about 3 cm long is made in the region of the fold of the right groin. The femoral artery is isolated from the nerve and the vein, then cleared and incised to introduce a polyethylene catheter, with inside diameter of 0.58 mm and outside diameter of 0.96 mm, filled with heparinized physiological serum. This catheter is advanced about 1 cm and makes it possible to take 50 mL of blood, collected in previously heparinized 20 mL syringes.
The wound is cleaned with iodinated polyvidone and the skin is sutured with polyglactine 2-0 thread (Vicryl, Ethicon). The animal is then returned to the animal house in the conditions described previously.
On an animal previously anaesthetized, supplementary local anaesthesia with Xylocaine (AstraZeneca, Monts, France) is applied, then an incision of about 4 cm is made in the neck, along the trachea. The right carotid artery is exposed. 300 U/mL of heparin sodium (Sanofi synthelabo, France) is administered intravenously just before fitting the vascular clamps (proximal and distal level).
After clamping the carotid, an arteriotomy (0.5 cm) is made proximally, at a distance of about 1 cm from the clamp, then distally, for inserting the vascular graft there by termino-lateral bypass. Anastomosis is performed by means of vascular threads 8-0. Once the graft is in place, the carotid artery is ligatured and blood circulation in the graft is verified.
The arterial substitutes are monitored for up to 3 months. The permeability of the substitutes is verified by Echo-Doppler. This apparatus measured the blood flow as well as the variation in diameter of the substitutes.
After the substitute has been in place for 1 and 12 weeks, the grafts and the control carotids (left) are removed, rinsed carefully with heparinized physiological saline solution, and then submitted to macroscopic and microscopic examination.
The animals are euthanized by injection of a lethal dose of pentobarbital sodium, according to the recommendations published by the European Commission (decree No. 2001-131 of 6 February 2001, linked to European Directive 86-609-EEC of 1986). The death of the animal is confirmed after respiratory and cardiac arrest.
Histological examination of the substitutes in
The observations with the scanning electron microscope (
The functionality of the arterial substitutes is monitored on a conscious animal by a non-invasive technique: “echo-Doppler”. This apparatus measured the velocity of the blood as well as the variation in diameter of the arterial substitutes.
The recordings obtained (
A leukocyte fraction from the peripheral circulation was obtained after density gradient separation. A mixture of heparinized blood and PBS (phosphate-buffered saline) (10 mL of blood in 16 mL of PBS) is added gradually to 10 mL of Histopaque® 1077 (Sigma, France), then centrifuged at 400 g for 30 min. The ring of leukocytes is aspirated with a sterile Pasteur pipette and transferred to a 50-mL tube containing 10 mL of MCDB 131 (Gibco, France) supplemented with 5 U/mL of heparin sodium (Sigma, France). The tube is then centrifuged at 250 g for 10 min, the supernatant is removed and the pellet is resuspended in 10 mL of heparinized MCDB 131. This last operation is repeated three times. The pellet is then resuspended in EBM-2 culture medium supplemented with a cocktail of growth factors (Single Quot®) (Clonetics, Belgium). About 1×106 EPC (endothelial progenitor cells) per cm2 are cultured in a 25 cm2 culture bottle treated with fibronectin (20 μg/mL) or a (PAH-PSS)3-PAH polyelectrolyte multilayer film. The culture medium is changed after four days, which makes it possible to remove the non-adherent cells, then every other day. These cells are cultivated for 2 weeks at 37° C. and 5% CO2.
Phenotypic characterization of the cells after culture for 14 days is carried out by observation in confocal laser scanning microscopy (
Semiquantitative investigation of fluorescence on images obtained in confocal microscopy after 14 days of culture (
EPCs derived from rabbit peripheral blood are recovered and the cells are counted on a Thoma cell. The viability is estimated according to the Trypan Blue exclusion test (Sigma, France). One volume of the final solution of Trypan blue is added to an equal volume of cellular suspension. The cells not allowing entry of the dye are considered to be alive.
The cellular suspension is adjusted and injected in the various matrices (arteries on which a monolayer of (PAH-PSS)3-PAH polyelectrolytes was deposited, and the control arteries) (with a length of 4 cm); the cell density is 1×107 cells/cm2. To allow better distribution of the cells, the endothelialized substitutes are placed in sterile Falcons, with gentle agitation for 4 hours.
The arteries are then put in culture bottles (one artery per bottle) and are put in an incubator at 37° C. (5% CO2 and 95% air). The culture time is one week.
To evaluate the retention of the EPCs sown in the lumen of the arteries (on which a polyelectrolyte multilayer film was deposited, and controls (without multilayer film)) and to improve their differentiation, the latter were exposed to laminar flow. The latter makes it possible to generate shear stresses of 0.1 to 0.25 Pa.
This study is carried out in a flow chamber developed in the laboratory (
A peristaltic pump permits circulation of the culture medium without growth factors. Upstream of the chamber, two syringes are added to the circuit, to create sinusoidal modulation in order to dampen the parasitic fluctuations of the flow. A gas mixture (5% CO2 and 95% air) is introduced in the reservoir of the medium to control the variations in pH. The system is put in a stove set to 37° C.
The shear stress is calculated from the following equation:
τ=4Qμ/πr3
τ: shear stress (Pa), μ: viscosity of the complete medium 0.9.10−3 Pa·s at 37° C.,
Consequently, knowing the value of the flow rate of the peristaltic pump, it is possible to find the shear stress exerted in the lumen of the artery. The peristaltic pump was calibrated by measuring the flow rate as a function of the rotary speed.
The flow rate of the peristaltic pump is calibrated by the graduation of the rotary speed (
y=0.0018 x
The retention of the mature endothelial cells on the substrate is evaluated by:
The culture substrates are glass slides:
The culture medium is as follows: alpha MEM+0.5% or 2% of SVF
Human mesenchymal stem cells are cultured at a seeding density of 5.103 cells per cm2.
The methods of differentiation into endothelial cells are:
The incubator is at 37° C., under 5% CO2.
The shear stresses in the flow chamber are 0.5 Pa, 1 Pa, 1.5 Pa, 2 Pa for 24 h, 48 h, 72 h or 96 h beginning at 7 days of culture (culture time after which the cells are confluent).
The quality of the differentiated endothelial cells can be verified:
The angiogenic potential can be evaluated by
| Number | Date | Country | Kind |
|---|---|---|---|
| 0704313 | Jun 2007 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2008/000832 | 6/16/2008 | WO | 00 | 9/3/2010 |
