WO2015124732A1 - Mélanges de polyisocyanopeptides fonctionnalisés par oligo(alkylène glycol) et de fibrine pour la culture de cellules - Google Patents

Mélanges de polyisocyanopeptides fonctionnalisés par oligo(alkylène glycol) et de fibrine pour la culture de cellules Download PDF

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WO2015124732A1
WO2015124732A1 PCT/EP2015/053633 EP2015053633W WO2015124732A1 WO 2015124732 A1 WO2015124732 A1 WO 2015124732A1 EP 2015053633 W EP2015053633 W EP 2015053633W WO 2015124732 A1 WO2015124732 A1 WO 2015124732A1
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fibrin
oligo
hydrogel
polyisocyanopeptide
cells
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PCT/EP2015/053633
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English (en)
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Egbert Oosterwijk
Wouter F.j. FEITZ
Petrus Johannes Geutjes
Alan Edward Rowan
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Stichting Katholieke Universiteit
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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33348—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing isocyanate group
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2210/00—Compositions for preparing hydrogels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/10—Applications used for bottles

Definitions

  • the invention relates to blends of oligo(alkyleneglycol) functionalized
  • polyisocyanopeptides for culturing cells for culturing cells, gels made from the polyisocyanopeptides, use of the gels and cell cultures prepared with the aid of the gels.
  • cell culture gel materials may be isolated from natural sources or completely synthetic. Gels such as collagen, which produce inherently lamellar structures, are incapable of forming complex 3D networks in isolation. Gels such as those derived from EHS mouse sarcoma cells resemble the extra cellular environment found in tissues much better than pure collagen and also provide three dimensional
  • Naturally derived gelators are difficult to fully characterise and require intensive batch to batch analysis to achieve this characterisation, biologically derived gels suffer from inherent variability, risk of contamination and pathogen transfer along with excessive price premiums. For many research groups, additional trace contamination such as unwanted growth factors inherently present in biologically sou reed materials are unacceptable experimental interferences and are unacceptable for use in-vivo.
  • synthetically derived gels such as those derived from poly(N-isopropylacrylamide) co-polymers exhibit low cell viability and cell differentiation ability, which requires additional mixtures of bioactives such as glucocorticoids and transforming growth factor beta (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor beta
  • Gelatable structures demonstrated above are not universal in nature and cannot be easily applied in a minimally invasive way in-vivo.
  • these materials suffer from the same drawbacks as described above resulting in poor cell viability.
  • Mechanically the properties of all of the biologically derived gels are dictated by the non-covalent interactions of the peptide subunits. The result is that the pore size and mechanical strength are relatively fixed. The mechanical properties and nature of the cross links are even more so fixed in the case of the synthetically derived gels.
  • WO 201 1/007012 discloses a hydrogel comprising oligo(alkylene glycol) functionalized polyisocyanopeptides.
  • the polyisocyanopeptides are prepared by functionalizing an isocyanopeptide with oligo-(alkylene glycol) side chains and subsequently polymerizing the oligo-alkylene glycol functionalized isocyanopeptides.
  • WO201 1/007012 suggests use of the hydrogels for tissue engineering or neuron regeneration.
  • WO201 1/00712 is hereby incorporated by reference
  • Fibrin (also called Factor la) is a fibrous, non-globular protein involved in the clotting of blood. Fibrin is currently widely used as a standard matrix material for growing cells. Fibrin is however from a biological nature, which means that the quality of the product may vary and is difficult to control. Moreover the price of fibrin is extremely high, which causes restrictions in the applicability of fibrin in tissue engineering.
  • the invention relates to a composition comprising fibrin and an oligo(alkylenglycol)- substituted polyisocyanopeptide.
  • the inventors have found that a blend of fibrin and an oligo(alkylenglycol)-substituted polyisocyanopeptide surprisingly support cell growth. This is surprising since the pure oligo(alkylenglycol)-substituted polyisocyanopeptide turned out not to support cell growth, while fibrin only supports cell growth when a high enough concentration and amount of fibrin is present. The inventors found that a substantial part of the fibrin can be replaced by the oligo(alkylenglycol)-substituted polyisocyanopeptide, while still generating a system that is viable for the growth and differentiation of stem cells.
  • the weight ratio of fibrin to the polyisocyanopeptide ranges between 5:95 and 99.5:0.5. More preferably the ratio is between 10:90 and 75:25, or between 15:85 and 50:50.
  • Oligo(alkyleneglycol)-substituted polyisocyanopeptide which are being used in the context of the present invention can be described with the following formula
  • methox tetra-ethyleneglycol substituted isocyanopeptide unit is:
  • composition may also comprise an oligo(alkyleneglycol)-substituted
  • copolyisocyanopeptide which is obtained by copolymerizing a first comonomer of an oligo(alkylene glycol) functionalized isocyanopeptide grafted with a cell adhesion factor and a second comonomer of a non-grafted oligo(alkylene glycol) functionalized isocyanopeptide.
  • the oligo(alkyleneglycol)-substituted copolyisocyanopeptide can be obtained by a process comprises the steps of:
  • step ii) adding a reactant of a spacer unit and a cell adhesion factor to the copolymer obtained by step i), wherein the spacer unit is represented by general formula A-L-B, wherein the linking group and group A are chosen to react and form a first coupling and the cell adhesion factor and group B are chosen to react and form a second coupling, wherein the first coupling and the second coupling are independently selected from the group consisting of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, oxanorbornadiene-based-azide couplings, vinylsulphone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin- strepavidin coupling, amine-carboxylic acid resulting in amides linkages, alcohol- carboxylic acid coupling resulting in esters linkages and
  • group L is a linear chain segment having 10-60 bonds between atoms selected from C, N, O and S in the main chain.
  • the cell adhesion factor supports the binding of cells to the gel.
  • the cell adhesion factor preferably is a sequence of amino acids.
  • amino acids that advantageously may be used in the present invention are N-protected Alanine, Arginine, Asparagines, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Thryptophan, Tyrosine, Valine.
  • Suitable sequences of amino acids include peptides such as RGD, GRGDS, IKVAV, KQAGDV and GRGDSP.
  • the cell adhesion factor may also be a growth factor such as VGEF and BFGF.
  • the cell adhesion factor may also be glycoproteins or mucins.
  • the composition contains thrombin.
  • Thrombin assists in converting soluble fibrinogen into insoluble strands of fibrin.
  • the invention also relates to a hydrogel comprising fibrin, oligo(alkyleneglycol)- substituted polyisocyanopeptide, between 70-99.9 wt% water, wherein the ratio between fibrin and the polysiocyanopeptide is between 5:95 and 95:5, preferably between 10:90 and 50:50.
  • the composition comprises water in an amount sufficient to form a gel which has a favourable mechanical strength for support of the cells.
  • the composition comprises water in an amount sufficient to form a gel which has a favourable mechanical strength for support of the cells.
  • the composition comprises water in an amount sufficient to form a gel which has a favourable mechanical strength for support of the cells.
  • composition comprises between 50 and 99.95 wt% water, preferably between 90 and 99.9 wt% water, more preferably between 95 and 99 wt% water.
  • a hydrogel is made from the copolymer and fibrin mixture as obtained by gelling with a suitable cell culture medium.
  • the hydrogel is a three dimensional hydrogel.
  • the polymer concentration in the hydrogel is preferably 0.1 -3.0 mg/mL, more preferably between 1 -3 mg/mL. If the polymer concentration in the hydrogel is too high, the hydrogel becomes too stiff for the cells to move and grow within the gel.
  • the hydrogel has an elastic modulus in the range 10-5000 Pa, preferably 100-1000 Pa at 35 °C as determined by plate - plate rheology experiments. This allows the cells to move and grow to form cellular network and 3D structures, like for example a prevascular system.
  • hydrogels obtained from the composition of the invention including the
  • oligo(alkylene glycol) polyisocyanopeptides differ from most of the previously reported polymer-based hydrogels in the highly structured nature of the network formed upon gelation.
  • the network consists of twisted bundles of laterally aggregated polymer chains. This arrangement is similar to the structure of the fibrilar networks that are formed upon the gelation of low molecular weight hydrogelators. It is supposed that this phenomenon is related to the high persistence length of the polyisocyanopeptides that favor an original mode of association. The association is triggered by the temperature induced modulation of oligo(alkylene glycol) side chains hydrophilicity which is a perfectly reversible phenomenon, resulting in a completely thermorevesible
  • Classical description of physical polymer hydrogels comprises the formation of an entanglement network chains in concentrated solutions, formation of a percolation network due to spinodal demixing, micro-crystallites formation, and formation of micelles network or lamellar structures which seemingly differ from the postulated association mode of the oligo(alkylene glycol) polyisocyanopeptide.
  • hydrogels resulting from the oligo(alkylene glycol) polyisocyanopeptide result from the lateral association of polymers fibers of about 5nm in diameter into larger twisted bundles that form the base of the polymeric hydrogel network. This results in a highly porous structure with pore size that can go down to 50nm in diameter.
  • the polymers used in the present invention present clear LCST transitions. For a given
  • oligo(alkylene glycol) polyisocyanopeptide this temperature can be modified by varying the ionic strength of the solution (salt effect) or more generally by the addition of any compounds able to modify the overall solvation state of the polymers.
  • the LCST of the materials can be further modulated by acting on the poly(isocyanide) backbone and namely on its conformation, with the use of acids or any compounds that can lead to conformational changes of the backbone helix.
  • Another way to modulate the LCST of the polymers is to co-polymerize monomers bearing different oligo(alkylene glycol) side chains. For example the polymerization of mixtures of tri- and tetra(ethylene glycol)isocyanodialanine in different ratio permitted to adjust the gellation temperature of the resulting copolymers between 22 °C and 60 °C in mQ water.
  • a further influence of polymers length has been observed in relation to the optical properties of the resulting gels. It was found that hydrogels prepared from chains with a lower degree of polymerization were prone to be turbid or opaque. Increasing the mean degree of polymerization resulted in a decrease of opacity of the hydrogels leading eventually to fully optically transparent materials.
  • the gel temperature may be adjusted to some extent, with the possibility to form stable structured gels at a temperature range of 5-50 °C, preferably 10-40 °C, more preferably between 15-35°C or between 20-25 °C, leading therefore to a new biomimetic matrix which can be used to encapsulate enzymes or cells and preserve their activity in vitro.
  • the polymers used in the invention appeared to have some interesting and
  • the gels in some cases were made up of 99.00 to 99.98% water. This means that there is only very little material required to generate a large volume.
  • a single wire of the polymer appeared to have a diameter of approximately 4 nanometer and a molecular weight of 2,500,000 Da.
  • the polydispersity index (PDI) was 1.6 and an average chain length varied between 500 nm - 2 micrometer.
  • the polymers appeared to be rather stiff, having a persistence length of 70 - 90 nm. It was also possible to obtain left and right-handed helices according to the peptide fragment chirality (optically active materials).
  • the polymers may therefore be used as a scaffold for biomolecules. We found that the porosity size is controlled by the concentration.
  • the cell culture according to the invention comprises the hydrogel as described above.
  • the cell culture is a three dimensional porous scaffold.
  • the invention further provides a process for making the cell culture according to the present invention, comprising the steps of: a) providing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, b) mixing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide with a cell culture medium and fibrinogen at a temperature below the gelation temperature of the hydrogel,
  • Cell culture medium can also be added after formation of the hydrogel.
  • the cell culture can in principle be made with any type of cell culture medium suitable for the culturing of (animal) cells. Suitable cell culture media support the growth and differentiation of the cells used in the method of the invention.
  • a cell culture medium for (mammalian) cells comprises salts, amino acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements, carbohydrates and other organic nutrients, dissolved in a buffered physiological saline solution.
  • salts include magnesium salts, for example MgCI 2 .6H 2 0, MgS0 4 and MgS0 4 .7H 2 0 iron salts, for example FeS0 4 .7H 2 0, potassium salts, for example KH 2 P0 4 , KCI; sodium salts, for example NaH 2 P0 4 , Na 2 HP0 4 and calcium salts, for example CaCI 2 .2H 2 0.
  • amino acids are all 20 known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine.
  • vitamins include: ascorbate, biotin, choline. CI, myo- inositol, D-panthothenate, riboflavin.
  • lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine.
  • detergents include Tween 80 and Pluronic F68.
  • An example of a buffer is HEPES.
  • growth factors/hormones/cytokines include IGF, hydrocortisone and (recombinant) insulin.
  • trace elements are known to the person skilled in the art and include Zn, Mg and Se.
  • carbohydrates include glucose, fructose, galactose, sucrose and pyruvate.
  • the culture medium may be supplemented with biomolecules. Examples of
  • biomolecules are biologicals, proteins, glycoproteins, peptides, sugars, carbohydrates, lipoproteins, lipids, glycolipids, silicas, drugs, nucleic acids, DNA, RNA, vitamins, nutrients, hydrolysates, polysaccharides, monosaccharides, recombinant peptides, mucins, enzymes, bioorganic compounds, recombinant biomolecules, antibodies, hormones, growth factors, receptors, contrast agents, cytokines, and fragments and modifications thereof.
  • suitable culture medium include Endothelial Growth Medium (EGM-2,
  • the optimal conditions under which the cells are cultured can easily be determined by the skilled person.
  • the pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen.
  • the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that these conditions optimal for the growth and productivity of the cells.
  • the person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity.
  • the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39°C, for example a temperature from 36 to 38°C, preferably a temperature of about 37°C; the optimal osmolarity between 260 and 400mOsm/kg.
  • the invention further provides a process for culturing cells, the process comprising the steps of: a) providing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, b) mixing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide with a cell culture medium, fibrinogen and cells, at a temperature below the gelation temperature of the hydrogel,
  • the present invention provides a cell culture comprising a hydrogel comprising the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, fibrin and at least one of endothelial cells and smooth muscle cells.
  • the cells are preferably co-cultured endothelial cells and smooth muscle cells.
  • the concentration of the cells may e.g. be 2,000 cells/mL to 1 ,000,000 cells/mL.
  • a 3-D structure, like for example a vascular system can be obtained thereby.
  • the invention further provides use of the cell culture according to the invention for growing tissue, for example for making a prevascular system.
  • the synthetic temperature-sensitive hydrogels have been synthesized from polyisocyanopeptides grafted with oligo(ethylene glycol) side chains.
  • polyisocyanopeptides where synthesized by polymerization of methoxy-functionalized monomers alone (synthesis of the homopolymer) or a mixture (with a varied ratio) of azide-functionalized monomer and a methoxy-functionalized monomer (synthesis of the copolymer).
  • Polymerization was catalysed by the addition of nickel(ll), Ni(CI204)2*6H20, with a molar ratio of 1 :10 000. This resulted in the polymerization of the methoxy and azide functionalized monomer in a random copolymer.
  • the statistical intermediate space between azide groups was varied stoichiometrically in case of the copolymer.
  • the (non)functionalized polymers were dissolved under semi-sterile conditions (laminar flow cabinet, Clean Air, Telstar, the Netherlands) in a 1 :1 mixture of smooth muscle cell medium (SMCM, sciencell, USA) and Vascular Cell Basal Medium (ATCC.USA) towards a final polymer concentration of 1.6, 2.0, 2.5 or 3.2 mg/ml (table 1 ). Before medium addition, the necessary amount of polymer was weighed (Mettler, AE2000) and exposed to ultraviolet light irradiation in the laminar flow cabinet for 5 minutes to eliminate potential pathogens.
  • SMCM smooth muscle cell medium
  • ATCC.USA Vascular Cell Basal Medium
  • the polymer was mixed with the sterile medium and stirred in a glass for a minimum of 24 hours at 4°C to ensure that the polymer was completely dissolved and swollen in the medium. 1 ml aliquots of the hydrogel were frozen at -20°C to prevent repeated freeze-thaw cycles and maintain hydrogel quality. Polymer quality controls
  • Circular Dichroism spectroscopic measurements were performed to confirm the hydrogen-bonded helical structure of the polymer backbone.
  • the polymer hydrogels were further analyzed by measuring the intrinsic viscosity (Molecular weigth) and rheology (C).
  • Molecular G' [Pa] 1.6 mg/ml
  • a sample (250 ul) of every PIC hydrogel batch was combined with 2 ml of Penicillin Streptomycin-free smooth muscle cell medium in a T25 flask (Corining, New York, USA) and kept in a humidified incubator containing 5% C02 at 37° C for several days to check for present pathogens.
  • Solidification tests were performed to ensure PIC hydrogel formation after injection of the PIC hydrogel in vivo.
  • PIC hydrogel aliquots (batch 28, DV9/10, batch 33 and DV1 1 , see table 1 ) were thawed for 5-10 minutes on room temperature and when thawed directly placed on ice (0 °C) for 30 minutes to reverse potential solidification processes in the aliquots.
  • 150 ul of every PIC hydrogel batch was either directly pipetted, or pushed through a pre-cooled 30G insulin needle (BD, United Kingdom), in a new and empty tube that was placed in a 37 °C water bath to mimic the in vivo situation.
  • the solidification time was measured and the different conditions were compared to each other by performing a PIC hydrogel inversion test on room temperature to evaluate any macroscopically visible differences between the pipetted and injected hydrogels.
  • PIC hydrogels used in the in vivo experiment were subjected, either alone or combined with fibrin (5:1 ratio), to an extra rheological analysis to explore potential differences in material properties between the injected and implanted scaffolds.
  • the collagen suspension was homogenized using a potter-elvehjem homogenizer (Louwers Glass and Ceramics Technologies, Hapert, The Netherlands) with an intermediate space of 0,35 mm.
  • the homogenized collagen suspension was centrifugated at 750 rpm for 30 minutes at 4 °C to remove entrapped air bubles and four ml of collagen suspension was poured into a 32 mm diameter polysterene mold (Greinier Bio-One, the Netherlands ) that served as the mold for the collagen scaffolds.
  • the plates were placed on an iron plate in the -20 freezer for at least 4 hours to allow homogenous freezing. Subsequently, the plates were transferred on dry ice (-80°C) towards the Zirbus lyophiliser (Zirbus Sublimator 500II, Bad Grund, Germany) and lyophilized for a period of two days.
  • MES 2-(N- morpholino)ethanesulphonic acid
  • a mixture of 33mM EDC and 6mM NHS in MES buffer was added for 4 hours at 21 °C to allow crosslinking.
  • Cross-linking was ended by two 1 hour washing steps with 0.1 M Na2HP04 in Milli-Q (ultra-pure water), followed by two washing steps in 1 M NaCI (Merck, Darmstads, Germany), six washing steps in 2M NaCI of which one overnight and six washing steps with Milli-Q to remove all salts from the constructs. All washing steps were performed under shaking conditions for 1 hour. After these washing steps, collagen scaffolds were frozen at -20 and again lyophilized for a period of two days as previously described.
  • MES 2-
  • scaffolds were packed per two in sterilization paper pouches and sended away for ⁇ -irradiation [25kGy, Synergy health, the Netherlands].
  • the morphology of the transverse section and air-and pan side of the 0.4% collagen scaffold was examined by a scanning electron microscope (SEM) (JSM 6310, JEOL Ltd, Tokyo). Dry samples of the non-crosslinked and cross-linked collagen scaffolds were fixed on a stub, using double-sided carbon tape, and sputtered with Gold for 60 sec by Scancoat Six SEM Sputter Coater (Temescal) before SEM analysis.
  • SEM scanning electron microscope
  • Morphology was observed with a magnification of 50x and 250x.
  • the average pore size was calculated using Image J (Fiji). TNBS analysis
  • the degree of collagen scaffold cross-linking was assessed by the determination of the amount of free amine groups spectrophotometrically using a 2,4,6-trinitrobenzene sulfonic acid (TNBS, Fluka, Buchs, Switserland) assay.
  • TNBS 2,4,6-trinitrobenzene sulfonic acid
  • Cross-linked and non- crosslinked (negative control) collagen pieces were weighted and incubated in 4% (w/v) NaHC03 (Merck, Darmstads, Germany) in Milli-Q at 21 °C for 30 minutes.
  • TNBS 0.5% (w/v) TNBS solution was added and incubated for 2 hours at 40 °C.
  • the calibration curve was made out of different concentrations of glycine (Scharlau Chemie, Barcelone, Spain).
  • the TNBS assay was performed three times. Fibrin
  • Fibrin was produced out of a fibrinogen solution (fibrinogen from bovine plasma, type 1 - S, Sigma-Aldrich, USA) with a concentration of 10 mg/ml sterile NaCL (0.9% NaCI, Verso I, France) and thrombin (Sigma-Aldrich, USA) with a concentration of 22 ul/ml.
  • a fibrinogen solution (fibrinogen from bovine plasma, type 1 - S, Sigma-Aldrich, USA) with a concentration of 10 mg/ml sterile NaCL (0.9% NaCI, Verso I, France) and thrombin (Sigma-Aldrich, USA) with a concentration of 22 ul/ml.
  • the fibrinogen solution was filtered before use using a 0.2 ⁇ filter (WhatmanTM, GE Healthcare Lide Sciences) under semi-sterile conditions (laminar flow cabinet, Clean Air, Tel star, the Netherlands).
  • HbSMCs Human Bladder Smooth Muscle Cells
  • PIC constructs with variable RGD conjugation concentrations (RGD 10 or RGD 22.5), polymer concentrations (1.0 mg/ml, 1.6 mg/ml, 2.0 mg/ml and 3.2mg/ml) and molecular weights (ranging between 400-700 kDa) were produced to evaluate cell behavior in time and select the best conditions for in vivo evaluation in view of construct preservation, cell survival, cell migration and branch-like structure formation.
  • PIC-fibrin hybrid constructs were produced with varying fibrin:PIC ratios (1 : 1 , 1 :5, 1 : 10 and 1 :50 (v/v)) to investigate if the hydrogel can serve as an inert environment for cell growth when it is mixed and functionalized with small amounts of fibrin.
  • non-functionalized PIC-fibrin scaffolds and PIC RGD 10-fibrin constructs were made (ratio 1 :5) to investigate the potential benefit of extra RGD conjugation in view of branch-like structure development.
  • Regular -non-functionalized (NF)-PIC and pure fibrin were included as a negative and positive control.
  • EBM-2 EBM-2, lonza
  • SMCM Smooth Muscle Cell Medium
  • polyisocyanopeptides A volume of 500 ⁇ of PIC having a concentration of 3,2 mg/ml was combined with a varying amount of fibrin (concentration of 10 mg/ml). The mixture was filled up with a 0,9% NaCI solution till a total volume of 1000 ⁇ was obtained. Cells were allowed to grow and the absence or presence of cell growth was investigated. Results are shown in table 2 and figure 3. Table 2

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Abstract

L'invention concerne une composition comprenant de la fibrine et un polyisocyanopeptide substitué par un oligo(alkylène glycol), des hydrogels contenant cette composition et des cultures de cellules à base de cette composition.
PCT/EP2015/053633 2014-02-21 2015-02-20 Mélanges de polyisocyanopeptides fonctionnalisés par oligo(alkylène glycol) et de fibrine pour la culture de cellules WO2015124732A1 (fr)

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WO2017037293A1 (fr) * 2015-09-03 2017-03-09 Stichting Katholieke Universiteit Procédé d'induction de la différenciation de cellules souches
WO2018067628A1 (fr) * 2016-10-05 2018-04-12 3M Innovative Properties Company Composition de fibrine comprenant un matériau support, procédé et objets pour plaies
EP3395861A1 (fr) * 2017-04-24 2018-10-31 Stichting Katholieke Universiteit Hydrogels biomimétiques à double réseau
CN109824885A (zh) * 2019-03-04 2019-05-31 南京理工大学 半胱氨酸改性的贻贝仿生组织粘接剂及其制备方法
US10940233B2 (en) 2016-10-05 2021-03-09 3M Innovative Properties Company Fibrinogen composition, method and wound articles

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CN109824885B (zh) * 2019-03-04 2021-03-26 南京理工大学 半胱氨酸改性的贻贝仿生组织粘接剂及其制备方法

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