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US-12624337-B2 - Integrated methods for precision manufacturing of tissue engineering scaffolds

US12624337B2US 12624337 B2US12624337 B2US 12624337B2US-12624337-B2

Abstract

Methods for the development and integration of multiple apparatuses and methods for achieving administration of stem cell therapies include precision manufacturing of tissue scaffolds and/or bioreactor substrates. The nano/microscale fiber material extrusion typifying the electrospinning process is married with the fiber alignment and layering characteristic of an additive manufacturing process. The method generates porous fibrous 3-D meshes with precision controlled structures from biopolymer melts and solutions and gels, blends, and suspensions with and without cells. A method of tracking the migration histories and shapes of stem cells on scaffold surfaces relies on immunofluorescent imaging and automated algorithms based on machine learning. The combination of the precision manufacturing method and the method of cell tracking and cell shape statistics, along with understanding of the intimate relationship between the cell shape/phenotype and scaffold architecture leads to an integrated method for cultivating and harvesting cells having desired phenotypes.

Inventors

  • Filippos Tourlomousis
  • Robert Chang
  • Dilhan Kalyon

Assignees

  • THE TRUSTEES OF THE STEVENS INSTITUTE OF TECHNOLOGY

Dates

Publication Date
20260512
Application Date
20210630

Claims (20)

  1. 1 . A melt electrospinning writing apparatus, comprising: a syringe barrel configured to house a printable material; a needle extending into said syringe barrel and comprising a needle tip configured to extrude printable material; a syringe pump for controlling the extrusion of printable material through said needle tip; a heating element for maintaining printable material in a molten state within said syringe barrel during extrusion; a grounded electrically conductive collector plate positioned a distance beneath said needle tip and configured for translational motion in at least two dimensions relative to said needle, said collector plate and said syringe pump being adapted to cooperate in the performance of an additive manufacturing process; a voltage source coupled to said needle tip and said collector plate for establishing an electrical potential between said needle tip and said collector plate sufficient to enable deposition of printable material onto said collector plate from said needle tip; and means for coordinating the operation of said syringe pump, said needle and said collector plate to deposit printable material with precision sufficient to form a 3-D printed biological scaffold with cellular-relevant geometrical feature sizes in three dimensions and a porous microarchitecture having a 0-90 degree or 0-45 degree pore structure provided by a plurality of woven porous substrates stacked on each other.
  2. 2 . The melt electrospinning writing apparatus according to claim 1 , further comprising a heat tunnel through which said syringe barrel extends, said heating element being positioned to deliver heat into said heat tunnel.
  3. 3 . The melt electrospinning writing apparatus according to claim 2 , wherein said heating element comprises a heat gun for delivering heated air into said heat tunnel and for heating a surface of said syringe barrel passing through said heat tunnel.
  4. 4 . The melt electrospinning writing apparatus according to claim 3 , wherein said heating element is configured to produce a temperature such that said surface of said syringe barrel has a temperature of about 77° C. to about 79° C.
  5. 5 . The melt electrospinning writing apparatus according to claim 4 , wherein said heating element is configured to produce a temperature such that said surface of said syringe barrel has a temperature of about 78° C.
  6. 6 . The melt electrospinning writing apparatus according to claim 3 , wherein said heating element is configured to produce a temperature such that said needle tip has a temperature of about 35° C. to about 45° C.
  7. 7 . The melt electrospinning writing apparatus according to claim 6 , wherein said heating element is configured to produce a temperature such that said needle tip has a temperature of about 40° C.
  8. 8 . The melt electrospinning writing apparatus according to claim 3 , wherein said heating element is configured to produce a temperature such that said collector plate has a temperature of about 25° C. to about 35° C.
  9. 9 . The melt electrospinning writing apparatus according to claim 8 , wherein said heating element is configured to produce a temperature such that said collector plate has a temperature of about 30° C.
  10. 10 . The melt electrospinning writing apparatus according to claim 1 , further comprising a thermal camera configured to monitor the temperature of said syringe barrel and said needle tip.
  11. 11 . The melt electrospinning writing apparatus of claim 1 , further comprising a vertical digital meter configured to monitor the distance between said needle tip and said collector plate.
  12. 12 . The melt electrospinning writing apparatus of claim 1 , wherein said collector plate is stationary.
  13. 13 . The melt electrospinning writing apparatus of claim 1 , wherein said geometrical feature sizes are 10 to 100 microns in length.
  14. 14 . The melt electrospinning writing apparatus of claim 1 , wherein said porous microarchitecture has geometrical feature sizes of about 100 microns or smaller.
  15. 15 . The melt electrospinning writing apparatus of claim 1 , wherein each of said woven porous substrates comprises a plurality of polycaprolactone fibers that are interwoven together.
  16. 16 . The melt electrospinning writing apparatus of claim 1 , wherein said means includes a programmable xy stage operatively connected to said collector plate and adapted to effect the translational motion of said collector plate relative to said needle such that said needle is stationary and said collector plate is movable relative thereto in at least two dimensions.
  17. 17 . A melt electrospinning writing apparatus, comprising: a syringe barrel configured to house a printable material having a polymeric composition; a needle extending into said syringe barrel and comprising a needle tip configured to extrude the printable material; a syringe pump for controlling the extrusion of the printable material through said needle tip; a heating element for maintaining the printable material in a molten state within said syringe barrel during extrusion from said needle tip as molten material; a grounded electrically conductive collector plate positioned a distance beneath said needle tip, said collector plate being operatively connected to a programmable motion-control system configured for translational motion in at least two orthogonal directions in coordination with said syringe pump to deposit the molten material along precise paths, thereby implementing an additive manufacturing process; and a voltage source coupled to said needle tip and said collector plate for establishing an electrical potential between said needle tip and said collector plate sufficient to enable deposition of the molten material onto said collector plate to form a 3D-printed biological scaffold comprising cellular-relevant geometrical feature sizes in three dimensions and a porous microarchitecture having a 0-90 degree or 0-45 degree pore structure provided by a plurality of woven porous substrates stacked on each other.
  18. 18 . The melt electrospinning writing apparatus of claim 17 , further comprising: a heat tunnel through which said syringe barrel extends, said heating element being positioned to deliver heat into said heat tunnel, and wherein said heating element comprises a heat gun for delivering heated air into said heat tunnel and for heating a surface of said syringe barrel passing through said heat tunnel.
  19. 19 . The melt electrospinning writing apparatus according to claim 17 , further comprising: a thermal camera configured to monitor the temperature of said syringe barrel and said needle tip; and a vertical digital meter configured to monitor the distance between said needle tip and said collector plate.
  20. 20 . A method for forming a 3-D printed biological scaffold comprising the steps of: extruding printable material from a syringe barrel through a needle having a needle tip configured to extrude the printable material; controlling the extrusion of the printable material through the needle tip; maintaining the printable material in a molten state within the syringe barrel during said extrusion step, wherein the printable material is extruded as a molten material; depositing the molten material from said needle tip onto a grounded electrically conductive collector plate positioned a distance beneath the needle tip, the collector plate being operatively connected to a programmable motion-control system configured for translational motion in at least two orthogonal directions whereby the molten material is deposited along precise paths; and establishing an electrical potential between the needle tip and the collector plate sufficient to enable deposition of the molten material onto the collector plate to form a 3-D printed biological scaffold with cellular-relevant geometrical feature sizes in three dimensions and a porous microarchitecture having a 0-90 degree or 0-45 degree pore structure provided by a plurality of woven porous substrates stacked on each other.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 15/998,685 filed Aug. 15, 2018, which claims priority to U.S. Provisional Patent Application Ser. No. 62/545,527 filed Aug. 15, 2017, the entire disclosures of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. CMMI-MME-1554150 awarded by the National Science Foundation. The government has certain rights in the invention. FIELD OF THE INVENTION The invention disclosed herein relates to the fields of additive manufacturing, tissue engineering, scaffold and bioreactor design and fabrication, regenerative medicine, stem cell expansion, stem cell differentiation, stem cell population homogeneity and heterogeneity, and automated image-based screening methods for the classification of stem cell phenotypes. BACKGROUND OF THE INVENTION The current understanding in stem cell expansion and differentiation, generally demonstrated on two-dimensional (“2-D”) substrates, is that it is necessary to change the stiffness of the substrate and/or include one or more bioactive reagents (typically cocktails) so that the phenotype of the stem cells can be conserved or modified during the expansion and differentiation of stem cells in regenerative therapies. Overall, the yields are relatively low and there are significant issues of maintaining the purity and homogeneity of the stem cell populations. The known methods for screening phenotypes of the stem cells (e.g., ELISA or flow cytometry) rely on bulk measurements and the use of relatively large stem cell populations. Thus, there are myriad issues in the current state of regenerative medicine using stem cells. SUMMARY OF THE INVENTION Embodiments of the invention disclosed herein include one or more methods that can be integrated to implement regenerative stem cell based therapies. The methods can be implemented under known paradigms, or may be integrated to implement new paradigms or approaches to regenerative stem cell based therapies. A first embodiment of the present invention includes a first method (“Method 1”) for precision manufacturing of three-dimensional (“3-D”) biomaterial scaffolds with precisely tunable porous microarchitectures and geometrical feature sizes at the cell's operating length scales (10-100 μm). This dimensional scale window of precisely controllable microscale geometrical feature sizes is unattainable with other polymer melt based additive manufacturing technologies such as, for example, fused deposition modeling (widely known as “3-D printing”). In embodiments of the method, high-fidelity fibrous scaffolds are fabricated through electrohydrodynamic (EHD) printing of a biopolymer melt using a melt electrowriting (“MEW”) technique. Embodiments of the method are used to generate porous fibrous 3-D scaffolds with precision-controlled porous microarchitectures from biopolymer melts. In exactly the same way, embodiments of the method can be used to generate 3-D scaffolds from a wide range of alternate materials such as polymer solutions, gels, blends and suspensions with and without cells. In embodiments, the scaffolds are, or are component parts of, devices such as static tissue engineered models and/or dynamic tissue engineered models embedded within perfusable bioreactors. A second embodiment of the present invention includes a second method (“Method 2”) for tailoring scaffold designs for stem cell expansion so that the stem cell phenotype is not altered. Embodiments of the method are used for homogeneous stem cell expansion without the presence of any cell-instructive chemicals and/or bioactive molecules and/or growth factors. A third embodiment of the present invention includes a third method (“Method 3”) for tailoring scaffold designs for stem cell expansion and subsequent targeted differentiation of the stem cell phenotype. Embodiments of the method are used for homogeneous stem cell expansion and subsequent targeted differentiation without the presence of any cell-instructive chemicals and/or bioactive molecules and/or growth factors. A fourth embodiment of the present invention includes a fourth method (“Method 4”) for machine learning based classification of stem cell phenotypes using methods (e.g., immunofluorescent imaging) for tuning manufacturing protocols for reproducible harvesting of targeted stem cell populations. This advanced manufacturing approach is biologically qualified with a metrology framework that models and classifies cell confinement states under various substrate dimensionalities and architectures. BRIEF DESCRIPTION OF FIGURES The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. For a more complete understanding of the present inven