WO2016080954A1 - Supports tridimensionnels de culture de cellules utilisant des suspensions de nanofibres de courte longueur et des substrats composites mixtes de nanofibres-microfibres - Google Patents

Supports tridimensionnels de culture de cellules utilisant des suspensions de nanofibres de courte longueur et des substrats composites mixtes de nanofibres-microfibres Download PDF

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WO2016080954A1
WO2016080954A1 PCT/US2014/065982 US2014065982W WO2016080954A1 WO 2016080954 A1 WO2016080954 A1 WO 2016080954A1 US 2014065982 W US2014065982 W US 2014065982W WO 2016080954 A1 WO2016080954 A1 WO 2016080954A1
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cell culture
nanofibers
nanofiber
cell
scaffold
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PCT/US2014/065982
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English (en)
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Miles C. Wright
Peter GEISEN
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Xanofi, Incorporated
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Priority to PCT/US2014/065982 priority Critical patent/WO2016080954A1/fr
Publication of WO2016080954A1 publication Critical patent/WO2016080954A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • 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
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose

Definitions

  • the present invention relates to creating 3 dimensional cell culture scaffolds using a wet-laid manufacturing process and containing a blend of short; staple nanoflbers and microflbers. More specifically, the invention relates to the use of a nonwoven process containing discrete-length polymeric nanoflbers for regulating or promoting tissue modeling, cell growth, function or gene expression as defined herein.
  • Fibers form, in part or in whole, a large variety of both consumer and industrial materials such as, for example, clothing and other textile materials, medical prostheses, construction materials and reinforcement materials, and barrier, filtration and absorbent materials.
  • materials such as, for example, clothing and other textile materials, medical prostheses, construction materials and reinforcement materials, and barrier, filtration and absorbent materials.
  • Nanoflbers are increasingly being investigated for use in various applications. Nanoflbers may attain a high surface area comparable with the finest nanoparticle powders, yet are fairly flexible, and retain one macroscopic dimension which makes them easy to handle, orient and organize. These materials have been constructed to closely represent the size and dimensions of fibers found in the native tissue extracellular matrix (ECM).
  • ECM native tissue extracellular matrix
  • die present invention relates to a cell culture scaffold which comprises a fabric substrate of cotton, synthetic or blend fibers containing wet laid polymeric, staple nanofibers of short cut lengths.
  • the staple polymeric nanofibers can be wet laid onto a fabric substrate of cotton, synthetic or blend fibers, or the nanofibers can be wet laid with other fibers to form a nonwoven substrate, or the nanofibers can be wet laid onto themselves to form a nonwoven containing only nanofibers.
  • the dried substrate can then be cut into discs and placed into individual culture wells or as a flat sheet cut to any size. Culture media and living cells can then be added onto the discs to give the cells a rigid substrate that contains randomly oriented nanofibers throughout the entire thickness of the substrate.
  • the nanofibers mixed with microfibers can be put into slurry form in cell culture medium and provide a structure that can be invaded and remodeled as part of the cell's normal tissue growth.
  • Figure 1 is a schematic of nanofibers and microfibers wet-laid into a composite substrate.
  • Figure 2 is a SEM image showing a cross-sectional edge view of a wet laid substrate comprised of 50% CA nanofibers (avg. diameter ⁇ 400 nm) and 50% Rayon microfibers (avg. diameter 9 urn).
  • Figure 3 Is a SEM image showing a top view of a wet laid substrate comprised of 50% PLA nanofibers (avg. diameter—400 nm) and 50% PET microfibers (avg. diameter 9 um).
  • Figure 4 is a SEM image showing a top view of wet laid substrates consisting of 10% cellulose acetate nanofiber/90% PET 1.5 denier microflber (A), 20% cellulose acetate nanofiber/80% PET 1.5 denier microflber (B), 30% cellulose acetate nanofiber/70% PET 1.5 denier microflber (C), 40% cellulose acetate nanofiber/60% PET 1.5 denier microflber (D).
  • Figure 5 is a SEM image showing a top view of a colony of MDA-MB-231 breast cancer cells attached to a microflber of a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ⁇ 400 nm).
  • Figure 6 is a SEM image showing a top view of a group of MDA-MB-231 breast cancer cells attached to the nanofibers of a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ⁇ 400 nm).
  • Figure 7 is an optical inverted fluorescent microscope image showing a top view of a group of green fluorescent protein (GFP)-expressIng MDA-MB-231 breast cancer cells attached to a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg, diameter ⁇ 400 nm).
  • GFP green fluorescent protein
  • Figure 8 Is a spinning disc confocal fluorescent microscope image of HepG2 cells grown for 5 days and stained with TRITC-phalloidin (red) showing actin filaments and Hoescht 33342 nuclear stain (blue) showing cell growth on die top (main image) and throughout the z-axls (top and right slices) of a substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter -400 nm).
  • Figure 9 is a spinning disc confocal fluorescent microscope image of NIH/3T3 cells grown for 5 days and stained with TRITC-phalloidin (red) showing actin filaments and Hoescht 33342 nuclear stain (blue) showing cell growth on the top (main image) and throughout the z-axis (top and right slices) of a substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ⁇ 400 nm).
  • Figure 10 Is a graph of cell proliferation data of MDA-MB-231 breast cancer cells grown over time on a substrate comprised of 70% PET microfibers (avg. diameter 13 um) and 30% PLA nanofibers (avg. diameter ⁇ 400 nm) (Cell Culture Scaffold 1) and 70% Rayon microfibers (avg diameter 11 um) and 30% CA nanofibers (avg. diameter ⁇ 400 nm) (Cell Culture Scaffold 2) In addition to 2D cell culture treated plastic
  • Figure 11 is a graph of cell proliferation data of NIH 3T3 mouse embroyonic fibroblast cells grown over time on a substrate comprised of 70% PET microfibers (avg. diameter 13 um) and 30% PLA nanofibers (avg diameter -400 nm) (Cell Culture Scaffold 1) and 70% Rayon microfibers (avg. diameter 11 um) and 30% CA nanofibers (avg. diameter ⁇ 400 nm) (Cell Culture Scaffold 2) in addition to 2D cell culture treated plastic
  • FIG. 12 shows the relative expression of matrix metalloproteInase-2 (MMP-2) mRNA in MDA-MB-231 breast cancer cells grown for 3 days.
  • MMP-2 matrix metalloproteInase-2
  • Figure 13 shows mRNA expression of primary human umbilical mesenchymal stem cells (MSCs) grown in a 0.5%wt slurry of PLA nanofibers in growth media and induced to differentiate into chondrocytes to create elastic cartilage.
  • MSCs primary human umbilical mesenchymal stem cells
  • D10 Collagen II to Collagen I expression
  • D10 day 10
  • nanofiber refers generally to an elongated fiber structure having an average diameter ranging from less than 50 nm -5 urn in some examples, in other examples ranging from less than 100 nm - 5 urn, and in other examples ranging from 200 nm -5 um.
  • the average diameter ranges from 40 nm -5 um, 40 nm - 2 um, 50 nm ⁇ 5 um, 50 nm -2 um, 100 nm - 5 um, 100 nm - 2 um, 200 nm -5 um, or 200 nm -2 um.
  • the "average" diameter may take into account not only that the diameters of individual nanofibers making up a plurality of nanofibers formed by implementing the presently disclosed method may vary somewhat, but also that the diameter of an individual nanofiber may not be uniform over its length in some implementations of the method.
  • the average length of the nanofibers may range from 100 nm or greater. In other examples, the average length may range from 100 nm to millions of nm.
  • the aspect ratio (length/diameter) of the nanofibers may range from 100 or greater. In other examples, the aspect ratio may range from 20 to millions. In some specific examples, we have demonstrated nanofibers with aspect ratios of at least 10,000. Insofar as the diameter of the nanofiber may be on the order of a few microns or less, for convenience the term "nanofiber” as used herein encompasses both nano-scale fibers and micro-scale fibers (mlcrofibers).
  • Polymers encompassed by the present disclosure generally may be any naturally- occurring or synthetic polymers capable of being fabricated into nanofibers.
  • examples of polymers include many high molecular weight (MW) solution-processable polymers such as polyethylene (more generally, various polyolefins), polystyrene, cellulose, cellulose acetate, poly(L-lactic add) (PLA), poly (lactic-co-gly colic acid) (PLGA), polyacrylonitrile (PAN), polyvinylidene difluoride (PVDF), conjugated organic semiconducting and conducting polymers, biopolymers such as polynucleotides (DNA) and polypeptides, etc.
  • MW high molecular weight
  • solution-processable polymers such as polyethylene (more generally, various polyolefins), polystyrene, cellulose, cellulose acetate, poly(L-lactic add) (PLA), poly (lactic-co-gly colic acid) (PLGA), polyacrylonitrile (P
  • Suitable polymers to form nanoflbers include vinyl polymers such as, but not limited to, cellulose acetate propionate, cellulose acetate butyrate, polyethylene, polypropylene, poh/(vlnyl chloride), polystyrene, polytetrafluoroethylene, poly(amethylstyrene), poly(acryllc add), poly(isobutylene), poly(acrylonitrile), poly(methacrylic acid), poly(methyl methacrylate), poly(l- pentene), poly(l,3-butadiene), poly(vinyl acetate), poly(2vinyl pyridine), 1,4-polyisoprene, and 3,4- polychloroprene.
  • vinyl polymers such as, but not limited to, cellulose acetate propionate, cellulose acetate butyrate, polyethylene, polypropylene, poh/(vlnyl chloride), polystyrene, polytetrafluoroethylene, poly
  • nonvinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(10-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(ll-undecanoamide), polyChexamethylene sebacamide), poly(m-phenylene terephthalate), poly(tetramethylene-m-benzenesulfonamide).
  • Additional polymers include those falling within one of the following polymer classes: polyolefin, polyether (including all epoxy resins, polyacetal, polyetheretherketone, polyetherimide, and
  • poly(phenylene oxide) polyamide (including polyureas), polyamideimide, polyaiylate,
  • polybenzimidazole polyester (Including polycarbonates), poh/urethane, polyimide, polyhydrazide, phenolic resins, polysilane, polyslloxane, polycarbodiimide, polylmlne, azo polymers, polysulflde, and polysulfone.
  • the polymer used to form nanoflbers can be synthetic or naturally- occurring.
  • natural polymers include, but are not limited to, polysaccharides and
  • derivatives thereof such as cellulosic polymers (e.g., cellulose and derivatives thereof as well as cellulose production byproducts such as lignin) and starch polymers (as well as other branched or nonlinear polymers, either naturally occurring or synthetic).
  • cellulosic polymers e.g., cellulose and derivatives thereof as well as cellulose production byproducts such as lignin
  • starch polymers as well as other branched or nonlinear polymers, either naturally occurring or synthetic.
  • Exemplary derivatives of starch and cellulose include various esters, ethers, and graft copolymers.
  • the polymer may be crossUnkable In the presence of a multifunctional crosslinldng agent or crossUnkable upon exposure to actinic radiation or other type of radiation.
  • the polymer may be homopolymers of any of the foregoing polymers, random copolymers, block copolymers, alternating copolymers, random tripolymers, block tripolymers, alternating tripolymers, derivatives thereof (e.g., graft copolymers, esters, or ethers thereof), and the like.
  • Microflber material type in the substrate may include but is not limited to PET, acrylic, aramids, Basofil, PVDF, nylon, PLA, polyethylene, PVA, Rayon, cellulose and cellulose derivatives.
  • the average diameter of the microfibers can be, for example, 2-500 micron, or 3-100 microns, or 5-50 microns, or 8-20 microns.
  • the average length of the microflbers can be, for example, 2-50 mm, or 3-30 mm, or 5-20 mm.
  • web is meant a fibrous material capable of being wound into a roll.
  • nonwoven web is meant a web of individual fibers or filaments which are Interlaid and positioned in a random manner to form a planar material without identifiable pattern, as opposed to a knitted or woven fabric
  • Nonwoven webs have been in the past formed by a variety of processes known to those skilled in the art such as, for example, meltblown, spunbound, wet-laid, dry-laid, and bonded carded web processes.
  • in vitro tissue culture is meant the ability to grow and multiply living cells out of the body in a controlled environment using culture media and synthetic materials.
  • a nonwoven or woven fabric substrate or web can be made from natural or synthetic fabrics and may be composed of fibers of cotton, cellulose, Lyocell, acetate, cellulose acetate, rayon, silk, wool, hemp, spandex (including LYCRA), polyolefins (polypropylene, polyethylene, etc), polyamide (nylon 6, nylon 6-6, etc), aramids (eg Kevlar®, TwaronQ, Nomex, etc), acrylic, or polyester
  • fabric blends fabrics of two or more types of fibers. Typically these blends are a combination of a natural fiber and a synthetic fiber, but can also include a blend of two natural fibers or two synthetic fibers.
  • Nanoflbers can be wet laid deposited onto a non-woven or woven substrate, which is placed on a filter mesh of 27-200 microns pore size.
  • Nanoflbers can also be deposited onto themselves without a substrate with basis weights ranging from 4 to 800 GSM or higher. In this case, longer length fibers provide substrate with enhanced Integrity and strength.
  • Polymeric nanoflbers can also be wet laid together with other nano-or microflbers to form a nonwoven substrate containing many types of fibers, including borosilicate glass fibers.
  • the XanoShear method of producing staple length polymer nanoflbers in a liquid shear process and adding them to a substrate by wet laying techniques is novel and has not been achieved in the prior art as nanofibers are typically produced as long f> 20 cm) dry fibers by electrospinning and meltblowing technologies.
  • the method of creating these discrete fibers is also different from the methods used to manufacture "fibrils" which are formed by phase separation or shear ripping from nanofibers or larger microfibers.
  • the diameter of a fibril is generally smaller than the diameter of the nanofiber with which it was associated, and typically smaller by an order of magnitude.
  • fibrils may also be characterized as nanofibers, in the present disclosure the term "fibrils" distinguishes these structures from the polymer nanofibers created using the XanoShear method.
  • the 3D cell culture scaffold described herein comprises a nanofiber-microfiber composite.
  • the nanofiber-micro fiber composite can be, for example, a wet-laid composite.
  • the nanofiber-microfiber composite can be, for example, a nonwoven composite.
  • the nanofiber-microfiber composite is not an electrospun or melt-blown composite.
  • the nanofiber-microfiber composite can comprise, for example, 5-95 wt% of microfibers and 5-95 wt% nanofibers, or 50-90 wt% of microfibers and 10-50 wt% nanofibers, or about 90 wt% of microfibers and about 10 wL% nanofibers, or about 80 wt.% of microfibers and about 20 wt% nanofibers, or about 70 wt% of microfibers and about 30 wt% nanofibers, or about 60 wt% of microfibers and about 40 wt% nanofibers, or about 50 wt% of microfibers and about 50 wt% nanofibers.
  • the nanofibers have an average length of 10-5000 microns, or 50- 2000 microns, or 100-1000 microns, or 200-800 microns. In some embodiment the nanofibers have an average fiber diameter of SO nm to 5 microns, or 100 nm to 1 micron, or 200-800 nm. In some embodiment; the nanofibers have an length to diameter (L:D) ratio of 20-20000, or 50-10000, or 200- 5000, or 500-2000.
  • the 3D cell culture scaffold described herein comprises PET microflbers and cellulose acetate nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of PET microflbers and cellulose acetate nanofibers.
  • the 3D cell culture scaffold described herein comprises PET microflbers and PLA nanofibers. In some embodiment, the 3D cell culture scaffold described herein comprises a nonwoven nanofi ber-m icroflber composite that comprises, consists essentially of, or consists of PET microflbers and PLA nanofibers.
  • the 3D cell culture scaffold described herein comprises Rayon microflbers and cellulose acetate nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of Rayon microflbers and cellulose acetate nanofibers.
  • the 3D cell culture scaffold described herein comprises Rayon microflbers and PLA nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of Rayon microflbers and PLA nanofibers.
  • the 3D cell culture scaffold described herein comprises a nonwoven nanofiber composite that comprises, consists essentially of, or consists of nanofibers wet- laid onto themselves.
  • the nonwoven nanofiber composite can consist essentially of or consist of wet- laid PLA and/or cellulose acetate.
  • a wet laid substrate was made consisting of 30% PLA nanofibers and 70% PET 1.5 denier microfibers (13 micron in diameter) or 30% cellulose acetate nanofibers and 70% Rayon 0.8 denier microfibers (11 micron in diameter) by weight
  • the substrate was dried and punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate.
  • the substrate was treated with 5x concentration of penicillln/streptomycin/amphotericin to remove any bacteria or fungus, and washed twice with buffered saline solution.
  • a wet laid substrate was made consisting of 50% polylactic acid nanofibers and 50% PET 1.5 denier microfibers (13 micron in diameter) by weight (Figure 3).
  • the substrate was dried and punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate.
  • the substrate was treated with 5x concentration of
  • NIH/3T3 or MDA-MB-231 cells were seeded into the wells at a density of 5000/cm 2 (4500 cells per well) in DMEM-high glucose containing 10% FBS and lx
  • the growth rate of cells was measured over a week using AlamarBlue assay (Life Technologies).
  • AlamarBlue assay (Life Technologies).
  • the NIH/3T3 and MDA-MB-231 cells grew exponentially, doubling in population every 24 hours.
  • Example 4 A wet laid substrate was made consisting of 30% polylactic acid nanoflbers and 70% PET 1.5 denier microfibers (13 micron in diameter) by weight The substrate was punched with a die into discs having a diameter of 13/8 inch and one disc was placed into each well of a 6-well plate. The substrate was treated with 5x concentration of penicillin/streptomycin/amphotericin to remove any bacteria or fungus, and washed twice with buffered saline solution.
  • MDA-MB-231 cells were seeded into either the scaffold wells or standard tissue culture treated plastic at a density of 20000/cm 2 (180000 cells per well) in DMEM-high glucose containing 10% FBS and lx penicillin/streptomycin. At days 2, 3, 4 and 7, RNA from separate wells in triplicate was isolated using TRIzol solution (Life Technologies). Gene expression of matrix metalloproteinase-2 (MMP-2) was analyzed by semiquantitative gel electrophoresis in multiplex using ribosomal 18s as a loading standard. MMP-2 is an indicator of extracellular matrix remodeling or migration by the cancer cell It was shown that initially the expression of MMP-2 in cells grown on the scaffold was increased 8 fold over traditional plastic and slowly decreased to a stable 2 fold expression by day 7 ( Figure 12).
  • a wet laid substrate was made consisting of 20% cellulose acetate nanoflbers and 80% PET 1.5 denier microfibers (13 micron in diameter) by weight
  • the substrate was punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate.
  • the plate was then treated with 19 kGy of gamma radiation to sterilize.
  • HepG2 cells were seeded Into the wells at a density of 15000/cm 2 (13500 per well). Cells were grown for 5 days in EMEM containing 10% FBS, Lrglutamine and lx penicillin/streptomycin.

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Abstract

La présente invention décrit un échafaudage tridimensionnel de culture de cellules, comprenant un mélange de microfibres et de nanofibres. L'invention décrit également une culture de cellules comprenant l'échafaudage de culture de cellules et comprenant en outre une cellule fixée à la nanofibre et un milieu de culture cellulaire en contact avec la cellule. La présente invention décrit également un procédé de fabrication de l'échafaudage de culture de cellules, comprenant l'application à l'état humide d'un mélange aqueux des microfibres et des nanofibres sur un substrat.
PCT/US2014/065982 2014-11-17 2014-11-17 Supports tridimensionnels de culture de cellules utilisant des suspensions de nanofibres de courte longueur et des substrats composites mixtes de nanofibres-microfibres WO2016080954A1 (fr)

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WO2006068809A2 (fr) * 2004-12-03 2006-06-29 New Jersey Institute Of Technology Reconnaissance de substrat par cellules souches mesenchymateuses humaines differenciables
CN102719391A (zh) * 2012-06-07 2012-10-10 江阴瑞康健生物医学科技有限公司 双相多孔三维细胞培养支架

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