WO2008140295A1 - Substrat de culture cellulaire, flacons de culture et procédés de culture cellulaire utilisant ledit substrat - Google Patents

Substrat de culture cellulaire, flacons de culture et procédés de culture cellulaire utilisant ledit substrat Download PDF

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Publication number
WO2008140295A1
WO2008140295A1 PCT/NL2007/050227 NL2007050227W WO2008140295A1 WO 2008140295 A1 WO2008140295 A1 WO 2008140295A1 NL 2007050227 W NL2007050227 W NL 2007050227W WO 2008140295 A1 WO2008140295 A1 WO 2008140295A1
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Prior art keywords
substrate
cells
protrusions
indentations
curvature
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PCT/NL2007/050227
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English (en)
Inventor
Jahr Holger
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Erasmus University Medical Center Rotterdam
Stichting Voor De Technische Wetenschappen
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Priority to PCT/NL2007/050227 priority Critical patent/WO2008140295A1/fr
Publication of WO2008140295A1 publication Critical patent/WO2008140295A1/fr

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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
    • C12N5/0068—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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • 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
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to cell culture equipment and to methods for cultivating cells.
  • the present invention relates to culture surfaces for use as attachment substrates in cell cultivation and to methods for preserving the cellular phenotype during cultivation, especially as associated with cell differentiation during tissue culture.
  • the present invention further relates to methods of preventing dedifferentiation of cells during tissue culture.
  • Tissue engineering is a method by which new living tissues are created in the laboratory to replace diseased or traumatised tissue.
  • Modern tissue engineering techniques involve the expansion of tissue-specific cells in vitro to regenerate the structural and functional tissue equivalents, and implantation of the expanded cells into the diseased or traumatised tissue.
  • the technique may be applied to a variety of cell types and tissues.
  • the tissue- specific cells may be either differentiated cells isolated from specific tissues, or undifferentiated progenitor cells (stem cells) induced to differentiate into a specific cell type. Suitable culture conditions for cell expansion are crucial in order to maintain or improve the potential of the cells to develop into the required tissue.
  • cartilage Unlike other tissues, cartilage has little ability to regenerate itself after trauma, disease or as a result of old age. This is due to the avascular nature of normal articular cartilage. Therefore, tissue engineering of cartilage is an area of considerable medical importance. In fact, the repair of damaged I
  • Tissue engineering based on in vitro cell expansion typically consists of harvesting chondrocytes from a cartilage biopsy, expanding the isolated cells in monolayer cultures until a sufficient
  • the monolayer cultivation step includes seeding of suspended cells in a culture medium that is contained in a glass or plastic plate (e.g. Petri dish) or flask.
  • a glass or plastic plate e.g. Petri dish
  • the plate or flask typically has a flat and smooth bottom to
  • the cells are incubated under conditions that allow for their expansion as a monolayer, whereby the plate's or flask's bottom surface serves as attachment substrate during development of the monolayer.
  • the phenotype of the cells also changes morphologically, in that the cells acquire a fibroblast-like appearance and flatten out. This phenomenon is termed dedifferentiation. Normal articular chondrocytes have a characteristic round shape and this phenotype is a prerequisite for proper extracellular matrix production, with the latter being
  • De-differentiated chondrocytes are not suitable for regenerative medicine applications as these cells have been shown to produce a fibro-cartilage with poor biomechanical properties.
  • chondrocytes can successfully be prevented by culturing chondrocytes under conditions of high cell density, in liquid suspension on microcarriers (beads) (preferably all of these together); in a collagen, agarose or alginate gel; on substrates with reduced adhesivity; or in the presence of actin disrupting agents. Essentially the success of these measures is attained in the presence of growth factors and other complex medium components. All these applications have specific drawbacks, like e.g. the biodegradability of the matrices, cell seeding problems, and cost aspects of growth factors, expensive medium components and/or the requirement for sophisticated dynamic culture reactors (spinner flasks).
  • TGF ⁇ transforming growth factor ⁇
  • TGF ⁇ transforming growth factor ⁇
  • the tissue that is eventually harvested is either in the form of a clump of cells surrounding a microbead core, or associated with a scaffold, which is a form not suitable for all applications wherein expanded cells are required.
  • the geometry of the substrate i.e. the surface structure of the attachment site for the cells
  • an attachment substrate that comprises protrusions that resemble convex micron-sized semispheres prevents dedifferentiation of cells grown on said surface.
  • an attachment substrate that comprises indentations that resemble concave micron-sized semispheres facilitates or augments differentiation of cells grown on said surface.
  • the surface geometry of an attachment surface may be used to control the differentiation potential of cells, and may be used to direct stem cell plasticity and transdifferentiation or preserve the phenotype - depending on choice of substrate.
  • the present invention in a first aspect provides a substrate for growth and attachment of cells, said substrate comprising protrusions or indentations having a surface curvature in at least one direction that is equivalent to a radius of curvature of between 1 and 500 ⁇ m and extending over an arc with an arc length subtending an angle of between 30 and 359 degrees.
  • the protrusion protrudes from the plane of the substrate, while the indentation protrudes beyond the plane of the substrate.
  • the distal surface of the protrusions and the opening of the indentations faces essentially parallel to the plane of the substrate.
  • the surface curvature of the protrusions or indentations may be convex (bulging outward), concave (hollow), or both (i.e. hyperbolic (e.g. saddle -like)).
  • the surface curvature of said protrusions or indentations extends in at least one direction so that the protrusion or indentation may have a surface in the form of (part of) a horizontal (spherical) cylinder (convex) or a (partly) horizontal (spherically) cylindrical impression (concave).
  • the surface curvature extends in more directions.
  • the surface curvature extends in all directions of the protrusion or indentation surface, such that said surface is at least partly spherical (convex or concave).
  • the protrusions comprise a convex surface curvature, while the indentations preferably comprise a concave surface curvature.
  • Even more preferred protrusions comprised on the substrate take the form of half spheres or semispheres.
  • the surface may also comprise a combination of protrusions and indentations as defined herein.
  • the protrusions or indentations may be situated close together on the substrate surface, their corners may touch each other so that they are connected, and they may even partly overlap. On the other hand, the protrusions or indentations may also be spaced apart so that they are separated and interspersed by sections of flat substrate surface.
  • the density of the protrusions or indentations per unit area of the substrate surface is not particularly limiting, although a higher density of protrusions or indentations will ultimately result in more cells per unit area of attachment substrate that benefit from the advantageous surface geometry, and thus in a higher density of non-dedifferentiated cells in case of a convex surface geometry, or a higher number of differentiated cells in the case of a concave surface geometry.
  • the protrusions or indentations are essentially part of the substrate as defined herein. They may be formed onto or in the substrate material by any method available, such as by casting, moulding or by pressing.
  • the substrate comprising the advantageous surface geometry may take any form.
  • tissue culture in conventional cell expansion protocols it may take the form of a 2D sheet or plate, for instance as a bottom side of a container for cell cultivation (flask, dish, plate, etc.) or it may take the form of a 3D scaffold structure capable of supporting three-dimensional tissue formation.
  • the present invention further provides a method for controlling cell differentiation during cultivation comprising culturing said cells on a substrate for growth and attachment of cells as defined above.
  • the present invention further provides a method for preventing dedifferentiation of cells, in particular chondrocytes, cultured under in vitro conditions, said method comprising culturing said cells on a substrate for growth and attachment of cells as defined above, wherein said substrate comprises a surface curvature as defined above that is convex.
  • the present invention further provides a method for augmenting differentiation of cells, said method comprising culturing said cells on a substrate for growth and attachment of cells as defined above, wherein said substrate comprises a surface curvature as defined above that is concave.
  • the present invention provides for a cell culture container comprising at least one wall, which under operating conditions is in contact with cultured cells, wherein said wall comprises a substrate as defined above.
  • the present invention provides for the use of a substrate for growth and attachment of cells, for controlling differentiation of cells in cell cultivation, said substrate comprising protrusions and/or indentations having a surface curvature in at least one direction that is equivalent to a radius of curvature of between 1 and 500 ⁇ m and extending over an arc with an arc length subtending an angle of between 30 and 359 degrees.
  • a substrate for growth and attachment of cells for controlling differentiation of cells in cell cultivation
  • said substrate comprising protrusions and/or indentations having a surface curvature in at least one direction that is equivalent to a radius of curvature of between 1 and 500 ⁇ m and extending over an arc with an arc length subtending an angle of between 30 and 359 degrees.
  • Preferred embodiments of the substrates used in aspects of the invention are as described in the aspect for the substrate itself.
  • Figure 1 shows various surface geometries suitable for application in aspects of the present invention.
  • Figure IA I refers in drawings A-H to various forms of protrusions from the substrate surface
  • Figure l.II refers in drawings A-H to various forms of indentations in the substrate surface.
  • 2D presentations may be projected in 3D space such as to form vertically symmetrical elements or they may extend parallel to the surface of the substrate for any length.
  • Short parallel extensions of Fig. IA I A, Fig. IA I B and Fig. IA I F will result in Fig. IB III C, Fig. IB III B, and Fig. IB III A, respectively, which take the form of cylindrical elements more or less "submerged" in the plane of the substrate.
  • Figure IB IV is a top view of close packed spheres forming the surface of the substrate.
  • Figure 2 shows a circle indicating the arc length s, radius r, and angle ⁇ as defined in the description below.
  • FIG. 3 shows images of P2 chondrocytes grown on two differently curved, but chemically identical substrates at day 7 in static culture.
  • (A) small, round-shaped articular chondocytes obtained on a multiple half- spherical shaped, fibrin-coated, solid silicon rubber moulded product with a microcarrier-like curvature (500 ⁇ m diameter type) are visible.
  • Chondrocytes from the same pre-culture (identical batch!) become large, flat and fibroblast- like in conventional, static monolayer culture (B) in regular 6-well plates. The latter is indicative of an unwanted de-differentiation towards fibroblast-like cells, while the former is a prerequisite for the production of the proper extracellular matrix being characteristic for healthy (hyaline) chondrocytes.
  • Figure 4 shows microscopically apparent cytoskeletal changes (actin staining) observed in chondrocytes isolated from microcarriers (A) and those cultured on a flat surface (B). These correlate well with the observed phenotype of cultured cells; i.e. compact and spherical when grown on curved surfaces (A, 500 ⁇ m type half-spherical fibrin-coated, silicon substrate) and stretched out on chemically identical, flat surfaces (B). Pictures were taken 72h after seeding.
  • Figure 5 shows a cartoon illustrating the key-steps of the experiment of Example 2: Human/bovine primary chondrocytes are isolated from tissue by enzymatic treatment (see below, “cells”) and pre-cultured (see below, “cell expansion culture”) until P2 in regular plates or flasks (flat bottom). PO cells (on left, bottom figure) may be used directly, but for most applications expansion cultures in order to increase cell numbers are desired The isolated cells are then dynamically seeded over night on a roller onto the differently shaped silicon moulded product in e.g. 50ml tubes.
  • Inner sides of the solid silicon rubber moulded products are fibrin-coated to facilitate cell attachment on inert rubber and either contain multiple half-spherical shaped protrusions with different radii of curvature, and or a flat surface mimicking regular culture substrates.
  • the "cell” in aspects of the present invention are preferably mammalian cells and include, but are not limited to, mammalian primary cells (i.e. differentiated and non-differentiated (so-called stem cells or progenitor cells)) and immortalized cells or cell-lines derived thereof; especially, but not exclusively, human, murine, equine, bovine, porcine cells of e.g., but not exclusively, mesenchymal or embryonic origin (i.e. in particular — but not exclusively, chondroblasts and progenitors thereof); osteoblasts, osteocytes, chondrocytes, smooth muscle cell (in particular vascular smooth muscle cells); fibroblasts, adipocytes, and stem cells or progenitors thereof.
  • mammalian primary cells i.e. differentiated and non-differentiated (so-called stem cells or progenitor cells)
  • immortalized cells or cell-lines derived thereof especially, but not exclusively, human, murine, equine, bo
  • chondrocytes refers to the material of which the surface functions as an attachment or adhesion site for cells.
  • the substrate may also be referred to herein as the attachment surface or the support.
  • the substrate surface in aspects of the present invention has a specific morphology, referred to herein as the "surface structure" or "surface geometry".
  • any material suitable for cell adhesion may be employed in aspects of the invention.
  • the material is non-toxic, biocompatible, and cell-culture approved.
  • bioplastics carbohydrates (e.g.
  • starch and cellulose starch and cellulose
  • proteins e.g. zein
  • polyesters polyhydroxyalkanoates
  • polylactic acids polyurethane
  • Solanyl-like materials polyethylene, polypropylene, polyethyleneterephtalate, polycarbonate, poly-3-hydroxybutyrate, polystyrene, polyglonic acid, polyvinyl and combinations and derivatives thereof.
  • the substrate may be (in the form of and/or part of) a container or (bio)reactor for cell cultivation, such as a vessel, a flask, a (Petri)dish, a bottle, a bag, a microtiter plate well.
  • Said part is preferably a wall, most preferably a bottom wall.
  • the sides of the reactor may be provided with the surface structure as defined herein.
  • culture reactors will be shaken, stirred, swirled or rocked in a horizontal plane in order to provide for sufficient aeration and nutrition of the cells, while the cells essentially stay on the bottom of the reactor.
  • the substrate may also be provided in the form of a sheet or disk, which may be used as an inlay in, for instance, a flask or dish to present the inside surface of said flask or dish with the substrate having the advantageous geometry.
  • a typical sheet or disk may have a thickness of from 0.001 to 3 cm.
  • surface curvature is defined as the rate of change of the slope of the tangent to the surface, or rate at which the surface deviates from its tangent plane, and is inversely related to the radius of the arc described by the osculating circle that defines said curvature in at least one direction.
  • the surface curvature may thus be quantitatively defined by the radius of curvature, a surface curvature defined by a radius of curvature of 500 ⁇ m being more flat or less curved, than a surface curvature defined by a radius of curvature of 50 ⁇ m.
  • the critical surface curvature is the minimal curve that a surface must attain in order to exert the desired effect of affecting differentiation of cells. The skilled person will understand that this may vary between cells and between cell-stage.
  • the surface area provided by a protrusion or indentation having a curved surface in at least one direction may further be defined by the "arc length" of the curvature.
  • the "arc length” (s) is the measure of the (non- Euclidian) distance along a curved line making up an arc which subtends (stretches under) an angle ( ⁇ ) in radians or degrees.
  • C (arc length s I radius r)
  • the arc length of a curve with a given radius may be defined by angle ( ⁇ ).
  • a central angle of 30 degrees thus refers to an arc that extends for a length of l/6* ⁇ * r, in one direction for a cylinder, and in all directions for a sphere.
  • curvature is responsible for the maintenance of a proper, round cellular shape of chondrocytes.
  • the critical (minimal) curvature i.e. the curvature that defines the turning point below which chondrocytes will adopt a fibroblast-like morphology
  • a surface curvature in at least one direction that is equivalent to a radius of curvature of between 1 and 500 ⁇ m and extending over an arc with an arc length subtending an angle of between 30 and 359 degrees is capable of preventing dedifferentiation in chondrocytes.
  • the radius of curvature is between 5 or 10 and 350 ⁇ m, more preferably between 25 and 250 ⁇ m, even more preferably between 50 and 125 ⁇ m.
  • the arc length subtends an angle of between from 30, 40, 50, 60, 80 or 90 to 120, 150 or 180 degrees. Arc lengths subtending even larger angles are possible, although very high angles may provide surfaces from which cells are increasingly difficult to recover without breaking the monolayer.
  • a protrusion may have a concave surface, effectively resulting in a substrate having the effect of concave surface geometry, whereas the elements essentially consist of protrusions.
  • indentations in the substrate surface may have a surface to which cells may become attached that is convex, effectively resulting in a substrate having the effect of convex surface geometry, whereas the elements essentially consist of indentations.
  • the protrusions may consist of spherical beads randomly or evenly distributed over the substrate surface, or closely packed together on the substrate surface so that each bead touches another and they may be configured in two or even three dimensional (multi layer) close packing, or in square array.
  • Microbeads glued to or otherwise attached or moulded into the surface of the substrate and having a diameter of 1-1000 ⁇ m (preferably 10-500 ⁇ m, or a diameter equivalent to a radius as defined above) are very suitable in aspects of the present invention.
  • curvature not seeding or culturing dynamics, is responsible for preventing dedifferentiation in chondrocyte cultivation, has important implications for cell cultivation in general.
  • Other cell types may be influenced in similar, or exactly opposite (convex surfaces), ways. Depending on the type of cell, the physiological needs may vary and for each type of cell there may be an optimal (culture substrate) surface curvature.
  • Cell cultivation in methods of the present invention may occur in the absence of specialized cultivation media such as serum-based media, and even in the absence of growth factors.
  • Incubation conditions will depend on the cell type cultured and these conditions as well as other cell cultivation issues are within the realm of the skilled artisan and may be optimised according to needs.
  • the cell cultivation surface of the present invention may have dimensions of from a few hundred microns to up to a few meters. Generally, surfaces in dimensions from 2 to 20 cm will support most tissue culture requirements.
  • the substrate surfaces may or may not be (enzymatically, (photo-) or electro-chemically) coated with macromolecular structures (polypeptides, polysaccharides, glycopeptides etc.) or any other coating to functionalize the surface or improve its properties.
  • the coatings may be applied to facilitate cellular attachment.
  • silicone rubber substrate material the material may be coated with fibronectin to render the material suitable for cell attachment.
  • BAC BAC were isolated from biopsy material by standard enzymatic digestion. Briefly, cartilage was sliced into small pieces and washed several times in physiological saline. A two-step digestion protocol was used. First, the cartilage was pre-incubated in pronase E (2mg/mL; Sigma, St Louis, MO) for 1.5 h, followed by overnight digestion in collagenase B (1.5mg/mL; Roche Diagnostics, Almere, The Netherlands). The chondrocytes were filtered using a 100- ⁇ m filter to remove small parts of undigested cartilage. Cell viability was tested using trypan blue staining and cells were counted.
  • Chondrocytes were seeded in culture flasks at a density of ⁇ 7,500 cells/cm 2 and subjected to standard differentiation culture. Typically, about 1.3xlO 6 cells are harvested from a regular T175 culture flask in expansion medium (Dulbecco's modified eagle medium; i.e. DMEM, Life Technologies, Breda, The Netherlands; Glutamax-I, supplemented with 10% fetal calf serum (FCS); gentamycine (50 mg/mL; Life Technologies), and fungizone (1.5 mg/mL; Life Technologies). Subconfluent chondrocytes were trypsinized at 80-90% visiual confluency, using Ix Trypsin-EDTA (0.25%
  • Cell culture on moulded silicon products Briefly, 500,000 cells are added to 50 ml of expansion medium (see cell expansion culture) in a 50 ml Falcon tube. Moulded silicon products (for details see silicon mould production) are then immersed in medium and the 50ml tube is placed on a roller inside a 37°C incubator for dynamic seeding. Cells are allowed to attach to the coated moulded product overnight (preculture) prior to transferring seeded moulded products to individual wells of a 6-well plate. These moulded products are immersed in 5 ml of medium (cell-side up) and incubated at 37°C with typical oxygenation settings. This process is also illustrated in Fig. 5.
  • Bovine chondrocyte i) monolayer (static) expanded (P2) and ii) spinner flasks expanded (dynamic seeding) cells were grown in spinner flask using two solid substrates.
  • a first substrate was microhex (Nalge Nunc International; Thermo Fisher Scientific Inc.; a shredded culture flask material consisting of essentially squared particles having flat surface) and a second substrate was biosilon beads/microcarriers (Krackeler Scientific, Inc., fully spherical material). Both materials were used in combination in one culture.
  • Shape, not seeding dynamics is responsible for the proper phenotype.
  • P2 chondrocytes are seeded dynamically (for details see Example 1 silicon mould cell culture), prior to a static (monolayer) culture period on "curved" and flat, but chemically identical, substrates (i.e. silicon rubber moulded products having curved and flat surfaces (see Fig. 5).
  • the experiment was performed as described in Example 1. Morphology was determined daily by microscopy. In static culture, we observed microscopically differences in cell morphology already within 48-hrs.
  • Fig. 3A round-shaped articular chondocytes are shown on a multiple half- spherical shaped moulded product, while chondrocytes from the same pre- culture became large, flat and fibroblast-like on the flat surfaces (Fig. 3B). The latter is indicative of an unwanted de-differentiation towards fibroblast-like cells, while the former is a prerequisite for the production of the proper extracellular matrix being characteristic for healthy (hyaline) chondrocytes.
  • the pictures are day 7 images.
  • Culture expanded chondrocytes tend to re-differentiate in alginate beads (a gel-like substrate, mimicking an artificial matrix) as shown by high collagen type II mRNA levels.. Without wishing to be bound by any theory, it may be the lack of 'grip' to a (flat) substrate that prevents cells from becoming "fibroblastic". On convex surfaces with a curvature above a certain minimal level, cells are relatively large compared to the part of relatively flat surface underneath them. The cell cannot easily stretch out on such surfaces and cannot become "fibroblast-like".
  • Example 4 Monolayer culture -curvature cut-off point determination

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Abstract

La présente invention concerne un substrat destiné à la croissance et à la fixation de cellules. Ledit substrat comprend des saillies et/ou des indentations présentant une courbure superficielle dans au moins une direction, équivalente à un rayon de courbure compris entre 1 et 500 μm et s'étendant sur une longueur arquée qui sous-tend un angle compris entre 30 et 359 degrés.
PCT/NL2007/050227 2007-05-16 2007-05-16 Substrat de culture cellulaire, flacons de culture et procédés de culture cellulaire utilisant ledit substrat WO2008140295A1 (fr)

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WO2002101028A2 (fr) * 2001-06-13 2002-12-19 The University Of Liverpool Substrats
EP1514920A1 (fr) * 2003-09-12 2005-03-16 Institut Curie Procédés et appareil pour le contrôle de l'organisation interne de cellules par adhésion cellulaire orientée

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