Cell and biofactor printable biopapers

Abstract

Disclosed herein is a structure having: a porous polymeric film permeated by a first extracellular matrix material; and a topcoat layer comprising a second extracellular matrix gel disposed on the film. Also disclosed herein is a method of: providing a porous polymeric film; permeating the film with a first extracellular matrix material; and applying a topcoat layer of a second extracellular matrix material to the film. Also disclosed herein is a method of: laser-machining holes through a film comprising collagen to form a web-like structure.

Claims

1. A method comprising: providing a plurality of structures each comprising: a porous polymeric film having pores permeated by a first extracellular matrix material; and a topcoat layer comprising a second extracellular matrix gel disposed on the film; placing living cells on or within each topcoat layer to form cell-seeded structures; and stacking the cell-seeded structures to form a stacked structure having alternating layers of polymeric film and topcoat layers; wherein at least one of the cell-seeded structures has cells positioned in a branched pattern before the cell-seeded structures are stacked.

2. The method of claim 1, wherein the cells are placed by a laser transfer method, laser-based printing, ink jet printing, micropen printing, syringe deposition, electrospray deposition, or a conformal printing method.

3. The method of claim 1, further comprising: incubating the stacked structure under conditions to induce cell differentiation or cell growth.

4. The method of claim 3, wherein the first or second extracellular matrix material comprises a growth factor.

5. The method of claim 1, wherein the living cells are placed in the branched pattern.

6. The method of claim 1, wherein the living cells are placed in a branched pattern comprising more than one type of cell.

7. The method of claim 1, wherein providing the structure comprises: providing the porous polymeric film; and permeating the film with the first extracellular matrix material; wherein the film is not cell-seeded.

8. The method of claim 1, wherein the branched pattern is formed by incubating the cell-seeded structures before stacking.

9. The method of claim 1, wherein the stacked structure comprises a vascular network that allows for flow of materials into and out of the stacked structure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) A more complete appreciation of the invention will be readily obtained by reference to the following Description of the Example Embodiments and the accompanying drawings.

(2) FIG. 1 shows (a) a fluorescent microscope image of live/dead stained HUVECs on a biopaper without topcoat and (b) the fluorescent live/dead image of the resulting HUVEC network on a thick Matrigel top coat

(3) FIG. 2(a) shows HUVECs printed to a Matrigel loaded biopaper in a stem and 45 branch pattern. FIG. 2(b) is a higher magnification image.

(4) FIG. 3 shows optical photographs of seeded papers in culture (a), individually (b), and successively stacked (c-f).

(5) FIG. 4 shows the maximum intensity projection of a 3D confocal microscopy scan of a live/dead stained construct

(6) FIG. 5 clear through imaging of a stacked construct.

(7) FIG. 6 shows examples of the polymer film: 6(a) shows a hexagonal arrangement of uniform holes and 6(b) shows a web-like structure.

(8) FIG. 7 schematically illustrates the printing and stacking process.

(9) FIG. 8 schematically illustrates a frame used with a collagen membrane and various neural cells.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

(10) In the following description, for purposes of explanation and not limitation, specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that the present subject matter may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known methods and devices are omitted so as to not obscure the present disclosure with unnecessary detail.

(11) Though there are many hurdles to creating engineered tissues and organs, two of the challenges are 1) sustaining the health and development of three-dimensional tissues, especially over 1000 m in thickness, via a vascular system; and 2) spatial patterning of heterogeneous tissues such as liver, kidney or brain tissues, or technically, any tissue type infiltrated by a vascular system containing vascular cells besides the primary tissue cell type.

(12) Disclosed herein is an approach to creating 3D tissues. The method uses thin polymer scaffolds (biopapers) as a mechanically stable sheets to be used in a cell printing apparatus. Each polymer sheet can be addressed with different growth factors and then loaded into a cell printer for patterned cell seeding. After printing, each sheet can be cultured to achieve the desired level of cell differentiation (e.g., vasculature formation) and/or tissue formation and then stacked into three dimensional structures. By printing multiple cell types in a defined pattern to each sheet, culturing, and then stacking the sheets, these biopapers can be used to enable heterogeneous tissue structures to be created in 3D. By printing vascular and microvascular cell types in defined network structures, the structure, size, and direction of 3D vasculature can be influenced in a way that allows for flow of oxygen, nutrients, and waste into and out of the tissue. This is in contrast to the wealth of work where microvasculature networks are seeded randomly in a 3D block matrix or scaffold, creating a random, non-flow appropriate network. Often this 3D scaffold block technique results in a dead zone in the center of the scaffold as there is no pre-existing vascular network to carry nutrients in or waste out. The stacked biopaper method allows vasculature to be built into each level of a 3D (stacked) scaffold. This approach can work without surface patterned chemical cues, or mechanical restrictions, so it also allows for spontaneous formation of the smallest microvasculature structures, and takes advantage of the natural developmental processes arising from cell-cell and cell-matrix interactions. Printable, stackable biopapers may enable the creation of thick, spatially patterned, vascularized 3D tissues and potentially organs.

(13) The biopapers are designed to 1) be used as a substrate for 2D cell printing; 2) enable handling and stacking to create a layered 3D construct; and 3) support the development of HUVEC networks while retaining fidelity of the printed pattern. Using a mold and a solvent casting/porogen leaching process, 200 m1 cm1 cm biocompatible PLGA scaffold sheets were created. Depending on whether cell infiltration or network formation was desired, the scaffold sheets could be impregnated with an ECM substance, Collagen Type I or Matrigel, respectively. In order to produce HUVEC networks which retained the printed structure, the surface gel thickness was increased to 30 m so as to decrease the mechanical influence of the relatively rigid PLGA scaffold on cell stretching. Four layer stacks of randomly seeded biopapers showed signs of interlayer merging and the feasibility for using these biopapers for layer-by-layer construction of 3D scaffolds. Biopaper substrates can be loaded with tissue specific hydrogels to support the desired cell type (here HUVEC). Biopapers enable the 2D cell printing techniques to be used to produce heterogeneously ordered 3D tissue constructs.

(14) The disclosed materials and methods may enable 2D printing techniques to be used to create three-dimensional biological tissue constructs comprised of multiple cell types with defined spatial arrangements.

(15) Stacking rigid scaffold sheets is an alternative to layering hydrogels and can be used to introduce polymer scaffolds without cytotoxic solvents. Described herein is the fabrication of thin polymer scaffold sheets (biopapers) for use in cell printing applications. Various biopapers have been made and used in a cell printer using biological laser printing (BioLP) (U.S. Pat. Nos. 7,294,367 and 7,381,440). Biopapers were patterned with high resolution (<30 m) 2D cell printing and then stacked to enable thick 3D tissue constructs to be created with well defined 3D cell arrangements. Besides mimicking the cell-cell arrangements of native tissue types, this approach could be particularly useful in prevascularization of engineered thick tissues. By stacking 200 m sheets prepared in this way, it may be possible to create anisotropic vascular networks with a spacing which resembles that of native tissue and mimics the structure/pattern of angiogenesis in the mammalian circulatory system. Towards this end cell printing onto hybrid biopapers that contain bilayers of PLGA and extracellular matrix (ECM) has been performed. Not only are these biopapers compatible with cell printing for forming patterns of cells, they also are stackable to form 3D cell constructs and support the formation of vascular networks.

(16) The polymeric film provides structural support for the biopaper during handling. The choice of polymer may depend upon the types of cells to be placed on it. Suitable polymers include, but are not limited to, poly(DL-lactide-co-glycolide) (PLGA) and collagen. PLGA is somewhat stiffer than collagen, such that collagen may be preferred for neural or brain cells.

(17) The polymer film is porous and may or may not be a non-woven material. The porous may be may be any means for creating empty regions in the film. The pores may be smaller than the thickness of the film or may be holes that traverse the entire thickness of the film. Suitable methods of forming the pores include, but are not limited to particulate leaching, solvent casting, and micromolding.

(18) Another method of forming the pores is by creating holes through the film by laser machining, collagenase digestion, molding, or other means. One possible configuration of the holes is a hexagonal arrangement of uniform holes. The holes may also be placed to form a web-like structure in the remaining film. Two web-like structures 10 are exemplified in FIG. 6. A web-like structure is defined herein as comprising a plurality of elongated radial members 15 radiating from a central region 20 and a plurality of first order crossing members 25 that are connected to adjacent radial members 15. Optionally, the structure may include second order crossing members 30 connected to adjacent first order crossing members 25, third order crossing members (not shown) connected to adjacent second order crossing members 30, etc. The crossing members may connect all possible pairings of adjacent members that they connect to, and may cross each other as in FIG. 6(b). The holes between the members may be small enough to support the extracellular matrix. The spacing between members may be, for example, 100-200 m. The film may be, for example, up to 30-50 m thick, and may be made from dry collagen such as that used to make a sausage casing.

(19) The first and second extracellular matrix may be the same or different materials, and may be in the form of a gel or hydrogel. Suitable material include, but are limited to, collagen, laminin, a mixture of collagen and laminin, hyaluronic acid, and any mixture thereof. Hydroxyapatite is also a suitable matrix for the topcoat for growth of bone cells. The matrix may optionally comprise biologically useful materials, including but not limited to a growth factor, a differentiation-inducing factor, an anti-apoptotic factor, a bio-active factor, or a synthetic peptide sequence. Matrigel is a commercially available material that includes collagen, laminin, and some of the factors named above. A given material may be used in different forms for different components of the structure. For example, a dry collagen film may be permeated and topcoated with a wet collagen gel or hydrogel.

(20) The total thickness of the structure is may make it suitable for handling during the application of cells to the structure without damage. The total thickness may be, for example, 50-500 m or 100-300 m. This thickness is divided between the film and the topcoat. When necessary from structural integrity, the film may compose a majority of the thickness with a thin top coat. A thinner film may be used when the film is placed within or on a rigid holding frame. Such a frame may comprise, for example, a metal such as aluminum for permanence, or a degradable polymer such as PLGAS when the frame will ultimately not be needed anymore. The frame may also comprise a non-degradable elastic polymer and/or have a microfluidic interface to the film. The interface can allow for the flow if nutrients and waste to and from the film. FIG. 8 schematically illustrates a frame used with a collagen membrane and various neural cells.

(21) The structure may comprise a plurality of living cells disposed on or within the topcoat layer to form a cell-seeded structure. These cells may include the cells originally placed on the structure and/or new cells grown or differentiated from the original cells. Any method of placing the cells that allows for at least some viable cells on the structure may be used. Suitable methods include, but are not limited to, a laser transfer method, laser-based printing, ink jet printing, micropen printing, syringe deposition, electrospray deposition, and a conformal printing method. One suitable laser method is BioLP, described in U.S. Pat. Nos. 7,294,367; 7,381,440; and 7,875,324. Other methods that may be applicable to the presently disclosed methods are described in Ringeisen et al., Jet-based methods to print living cells, Biotechnol. J. 1, 930-948 (2006); Ringeisen et al., Biomaterials 23, 161-166 (2002); Ringeisen et al., Tissue Eng 10 (3-4): 483-491 (2004); Chen et al., App. Surf. Sci. 252, 8641-8645 (2006); Barron et al., Annals of Biomed Engineering, 33 (2): 121-130 (2005); Wu et al., BioLP Printing and Development of HUVEC and HUVSMC Branch/Stem Structure on Hydrogel Layers, Biofabrication 2 (2010) 014111; and U.S. Pat. Nos. 6,177,151; 6,805,918; 6,815,015; 6,905,738; and 6,936,311.

(22) The cells may be placed in random or random-like arrangements, in an anisotropic arrangement, or both. Anisotropic arrangement may be useful for vascular cells to enable the formation of a vascular network throughout the structure. Any other type of cell may be used including, but not limited to, neural cells, endothelial cells, osteoblasts, neurons, astrocytes, and hepatocytes. Any cells capable of growth and/or differentiation may also be used.

(23) A plurality of the structures with cells may be stacked to form a stacked structure having alternating layers of polymeric film and topcoat layers. The stacked structure may be incubated under conditions to induce cell differentiation or cell growth, including the use of growth factors. This method may be used to form a three-dimensional tissue. Incubation may also occur in individual layers that may be stacked afterward. FIG. 7 schematically illustrates the printing and stacking process. Such a structure may be useful as a brain tissue simulant for testing ballistic damage.

(24) The following examples are given to illustrate specific applications. These specific examples are not intended to limit the scope of the disclosure in this application.

Example 1 (Comparative)

(25) Polymer Film without a Topcoat

(26) Biopaper sheets were fabricated using a solvent casting/particulate leaching method with 3.6% w/v Poly(DL-lactide-co-glycolide) M.sub.w 40,000-75,000, lactide:glycolide (65:35) Lactel (Sigma #BP-0200) in chloroform with pores formed by +80 MESH (177 m) NaCl powder (GFS Chemicals, Inc.). The PLGA scaffolds were cast in rectangular molds formed by 1 mm thick polydimethylsiloxane (PDMS) placed on glass. The NaCl porogen was leached with at least 6 serial rinses in DI water for 1 hour each. Once rinsed the air dried over night the PDMS mold was peeled away from the glass leaving the PLGA scaffold strips on the glass. A sharp razor blade was used to separate the scaffold strips from the glass and they were rinsed once more. After drying again the strips of scaffold were cut into 11 cm.sup.2 squares. Two days prior to use, the PLGA squares were covered with a stainless steel mesh and submerged in basal medium under a vacuum to pre-wet them and remove air bubbles.

(27) For 3D cell culture experiments, Collagen Type I from rat tail (BD Biosciences 3.67 mg/mL) was brought to alkalinity with 1M NaOH and mixed with an equal amount of cell suspension (510.sup.6 cells/mL) yielding a collagen density of 1.835 mg/mL. Wetted scaffolds were removed from media and placed in a Petri dish. Excess medium was removed from the paper with a Pasture pipette placed on the dish bottom next to the papers edge. The paper was infused with cells and collagen by pipetting 50-100 L of collagen/cell suspension onto the surface and any excess suspension which seeped out was gently removed with the micropipetter. The papers were then placed in the incubator for 45 min to allow the collagen to gel and cells to attach. After initial seeding, a sterile stainless steel mesh was placed over the biopaper and medium was added. Media was changed after 24 hours and then every 2 days.

Example 2

(28) Polymer Film with a Topcoat

(29) Some experiments involved printing or randomly culturing human umbilical vein endothelial cells (HUVECs) on the surface of biopapers with the expectation of network formation. For these experiments, Matrigel (BD Biosciences) was used as the ECM hydrogel. Unseeded scaffold sheets were removed from the wetting medium and placed onto 22 mm.sup.2 glass coverslips in 35 mm Petri dishes and then cooled in a 4 C. refrigerator. Matrigel was thawed and kept on ice. Petri dishes containing PLGA scaffolds were then placed on ice. Excess media was removed from the scaffolds with a Pasteur pipette and 50-100 L Matrigel was then micropipetted onto the scaffolds. The biopapers were then allowed to gel in a humidified incubator at 37 C. for 1 hour. After being gelled these scaffolds were considered as biopapers without a topcoat. To create biopapers with a 30 m topcoat, an additional 30 L of Matrigel was pipetted to the surface of the biopaper and placed back into the incubator to gel again. The 30 m thickness was estimated by the volume of Matrigel divided by the paper area and checked by measuring the Z distance between focus on pore surface and gel surface using confocal microscopy.

(30) Test 1: Ability of Paper to Support Cell Differentiation:

(31) HUVECs were seeded on Matrigel-loaded biopapers with and without an additional 30 m top coat of Matrigel to determine whether typical network morphology developed. FIG. 1(a) shows a fluorescent microscope image of live/dead stained HUVECs on a biopaper without topcoat. FIG. 1(b) shows the fluorescent live/dead image of the resulting HUVEC network on a thick Matrigel top coat. The live/dead stain shows that nearly 100% of the seeded cells remain viable on biopapers with and without a Matrigel topcoat after 24 hours of culture. However, there is a noticeable difference in the extent to which the HUVEC network morphology adheres to the PLGA pore morphology. Without the 30 m topcoat, the HUVECs form networks along the rigid boundaries of the scaffold.

(32) Test 2: Ability of Paper to Support BioLP Printing and Retain Fidelity of Printed Cell Structures:

(33) A typical result of HUVECs printed to a Matrigel loaded biopaper in a stem and 45 branch pattern is shown in FIGS. 2(a) and (b). The live/dead stain indicates that greater than 95% of the printed cells are viable on the biopapers after 24 hours of culture. FIG. 2(b) is a higher magnification image of the printed HUVEC structure. Many of the printed HUVECs have begun linking along the length of the branched pattern (dashed line shows original stem/branch printed pattern).

(34) Test 3: Stack Ability of Papers, Interlayer Interaction:

(35) Individual biopapers were randomly seeded throughout with HUVECs in collagen for 1 day prior to stacking. FIG. 3 shows optical photographs of the seeded papers in culture (a), individually (b), and successively stacked (c-f). The stacked construct was held down with a stainless steel mesh and cultured for 4 more days. FIG. 4 shows the maximum intensity projection of a 3D confocal microscopy scan of the live/dead stained construct. Because of the opacity and diffraction of the PLGA papers, clear through imaging of the stacked construct was performed at scattered sites in the XY plane of the biopaper where several pores were aligned to form a clear through hole or a nearly clear-through hole in the scaffolding (FIG. 5). After 5 days of culturing, the construct had compressed to a total thickness of about 400 m. 3D confocal microscopy of stacked biopapers at 5 days showed a cell density of 1.310.sup.6 cells/cm.sup.3. Of these cells, live/dead staining showed a viability of less 25%.

(36) Obviously, many modifications and variations are possible in light of the above teachings. It is therefore to be understood that the claimed subject matter may be practiced otherwise than as specifically described. Any reference to claim elements in the singular, e.g., using the articles a, an, the, or said is not construed as limiting the element to the singular.