Patent Application
20210369917
This invention relates to modular biofabrication platform for diverse tissue engineering applications. More particularly, this invention is directed to biomanufacturing enabled by bioprinting.
Volumetric muscle loss (VML) resulting from traumatic injury and disease, and VML-like congenital and genetic conditions such as cleft lip/palate, are common in both the military and civilian populations1,2. By definition, such injuries and conditions exceed the considerable endogenous regenerative capacity of skeletal muscle and result in permanent cosmetic and functional deficits2. Despite significant advances in surgical procedures, VML treatment often requires multiple surgical interventions with generally poor cosmetic and functional outcomes, as current treatments for VML and craniofacial defects do not significantly promote regeneration of missing muscle tissue. Surgical treatments include skin grafts and autologous muscle flaps2. Utilizing autologous muscle from the patient poses the risk of donor site morbidity and relies on the availability of sufficient muscle for transfer3,4. Furthermore, there is the possibility of muscle flap failure3. Unfortunately, the most devastating and persistent cosmetic and functional deficits resulting from traumatic VML in service members and civilians cannot be solved with existing reconstructive procedures, and are a major source of long-term disability1.
With a high occurrence of VML injuries and a lack of treatment options that address the incurred functional and cosmetic deficits, there is a clinical need for additional therapies. There is no current standard of care for VML injury that yields satisfactory functional outcomes, nor any biologic or combination product (of which the present inventor is aware of) that has received Food and Drug Administration (FDA) approval for VML repair. Despite some encouraging initial clinical results from implantation of decellularized extracellular matrices (dECM) for treatment of VML in patients, it is clear that there is still significant room for therapeutic improvement5-7. As such, continued development of tissue engineering and regenerative medicine technologies/products has enormous potential to provide a therapeutic solution for VML, and this opportunity has spurred robust preclinical activity8-15. Finally, as discussed in great detail herein, there are still significant challenges remaining with respect to biomanufacturing these products.
Decellularized Extracellular Matrix (dECM): A “Ready-Made” Scaffolding Material
Methods that have been employed for skeletal muscle repair include implantation of acellular scaffolds16-22, minced muscle grafts23, cell-laden scaffolds11-13,24-26, and bioprinted constructs27. While a variety of naturally derived and chemically synthesized scaffolding materials have been explored, one of the more promising materials for clinical translation is dECM, which will be one of the aspects of the various embodiments of the present invention. The extracellular matrices (ECM) is critical to tissue structure and function and plays an important role in cell signaling for migration, proliferation, and differentiation28-31. However, natural ECM has an extremely complex structure of proteins (such as collagen, laminin, and fibronectin) and polysaccharides (particularly glycosaminoglycans, or GAGs, such as hyaluronic acid)28,29,32,33. This complex structure of ECM is difficult to mimic in engineering29, and the use of biologically sourced materials provides an effective method for capturing this complexity. The field of tissue engineering has made significant strides in harnessing the inherent complexity of the ECM itself by establishing methods for using dECM in tissue regeneration32-36. The promise of dECM as a material in tissue engineering extends beyond its complex structure and there is evidence that dECM retains bound growth factors such as vascular endothelial growth factor (VEGF) which could be beneficial in the context of tissue regeneration30,37,38. Importantly, several sources of dECM have been FDA-approved as implantable devices, including porcine small intestine submucosa (SIS), porcine urinary bladder, human/porcine/bovine dermis, and porcine heart valves29,32,34. Additionally, these materials are abundantly available from porcine sources and fortunately do not illicit a harmful immune response, as components of the ECM are highly conserved across species39.
In the context of preclinical studies for repair of VML, decellularized ECM derived directly from skeletal muscle explants has also been evaluated18,22. However, regardless of the origin of the dECM, while acellular repairs can restore aspects of muscle volume and morphology, they do not promote appreciable muscle fiber regeneration when compared to scaffolds that include a cellular component. This is true even when some functional recovery is observed, and demonstrates the importance of including a cellular component13,23,24. Current methods of combining cells and dECM in a therapy to create a microenvironment that is more favorable for endogenous skeletal muscle regeneration and functional recovery after VML injury have been explored20,24,26. One such current method is described below.
Tissued engineered muscle repair (TEMR) is an autologous implantable construct capable of volume reconstitution and restoration of clinically relevant force/tension following VML injury in biologically relevant rodent models8,9,11-15,26. The manual biomanufacturing process for the TEMR construct has been published11-14, and is shown generally in the top portion of
Briefly, the current TEMR construct is created by seeding approximately 1×106 muscle progenitor cells (MPCs)/cm2 onto each side of a BAM scaffold, followed by 10 days of cell proliferation and differentiation, and then 5-7 days of bioreactor preconditioning, in vitro (i.e., 10% cyclic mechanical stretch, 3 times per minute for the first 5 minutes of every hour). Following this conditioning and maturation period, the TEMR construct exhibits a largely differentiated cellular morphology consisting primarily of myoblasts and myotubes. The entire manual TEMR manufacturing process takes 12 days prior to bioreactor preconditioning, as follows: 2 days of manual seeding (1 day per side); 3 days proliferation and 7 days of differentiation. Implantation of TEMR at the site of VML injury in the present inventor's biologically relevant rodent models can restore clinically relevant force/tension (60-90% functional recovery) within 2-3 months of implantation, providing important proof of concept11-15,26. The present inventor's most recent publication indicates that the size of the injuries envisioned as currently amenable to treatment via TEMR implantation (≈2 cm2) scale well to the present inventor's currently proposed indication for secondary revision of unilateral cleft lip in patients26. With current approaches, sufficient autologous cells for creation of the TEMR construct for this purpose can likely be obtained from a biopsy of ≈1000 mg, perhaps less, of donor leg muscle. Of note, such constructs would also be applicable to the repair of some muscles in the hand and shoulder.
An aspect of an embodiment of the present invention bioprinting methods and related systems hold promise for addressing biomanufacturing challenges associated with scale-up for clinical translation of this technology. These challenges extend beyond the context of VML and the TEMR construct specifically, but the TEMR will be used for the purpose as a model to illustrate these points.
As mentioned above, the following patents, patent applications and patent application publications as listed below are related to aspects of embodiments of the present invention and are hereby incorporated by reference in their entirety herein. The bioreactor related systems, bioreactor related devices, bioreactor methods, bioreactor controllers, methods for bioreactor controllers, and non-transitory computer readable medium to execute a method for a bioreactor controller are considered part of the present invention, and may be employed within the context of the invention.
a. International Patent Application Serial No. PCT/US2016/051948, entitled “BIOREACTOR AND RESEEDING CHAMBER SYSTEM AND RELATED METHODS THEREOF”, filed Sep. 15, 2016; Publication No. WO 2017/048961, Mar. 23, 2017; the disclosure of which is hereby incorporated by reference herein in its entirety.
b. U.S. Utility patent application Ser. No. 15/760,009, entitled “BIOREACTOR AND RESEEDING CHAMBER SYSTEM AND RELATED METHODS THEREOF”, filed Mar. 14, 2018; Publication No. US-2018-0265831-A1, Sep. 20, 2018.
c. International Patent Application Serial No. PCT/US2017/045299, entitled “BIOREACTOR CONTROLLER DEVICE AND RELATED METHOD THEREOF”, filed Aug. 3, 2017; Publication No. WO 2018/027033, Feb. 8, 2018; the disclosure of which is hereby incorporated by reference herein in its entirety.
d. U.S. Utility patent application Ser. No. 16/322,691 to Christ, et al, “Bioreactor Controller Device and Related Method Thereof”, Feb. 1, 2019.
An aspect of an embodiment of the present invention provides, among other things, a bioprinting method, wherein the method may comprise: disposing a scaffold onto a bioassembly device; disposing said bioassembly device, with said scaffold, onto a bioprinter; bioprinting onto a first side of said scaffold or both said first side and a second side of said scaffold, which is disposed on said bioassembly device that is disposed on said bioprinter; transferring said bioprinted scaffold, which is disposed on said bioassembly device, onto a bioreactor; and creating tissue engineered construct while said bioprinted scaffold remains on said bioassembly device and in said bioreactor.
An aspect of an embodiment of the present invention provides, among other things, a bioassembly device for use with a bioprinter, wherein said device may comprise: a top portion and a bottom portion that are configured to secure a scaffold there between while said bioprinter performs bioprinting onto a first side of said scaffold or both said first side and a second side of said scaffold.
An aspect of an embodiment of the present invention provides, among other things, a bioprinting system, where the system may comprise: a designated area configured for receiving a bioassembly device, which includes a scaffold disposed in said bioassembly device; and a print head configured for bioprinting onto a first side of said scaffold or both said first side and a second side of said scaffold, while said bioassembly device is in said designated area of said bioprinting system.
An aspect of an embodiment of the present invention provides, among other things, a system, method, and computer readable medium of bioprinting that is used to enable automated fabrication of various constructs with high reproducibility and scalability, while reducing costs and production timelines. The bioprinting applications provides a critical component to the further enrichment the overall biomanufacturing paradigm. The biofabrication of sheet-like implantable constructs and other construct types and geometrical structures with cells deposited on both sides—a process that may be both scaffold and cell type agnostic, and furthermore, is amenable to many additional tissue engineering applications beyond skeletal muscle.
An aspect of an embodiment of the present invention provides, among other things, bioprinting on sheet-based scaffolds applied to the creation of implantable tissue engineered constructs with potentially diverse clinical applications. As a non-limiting example, tissue engineered muscle repair (TEMR) provides an aspect of an embodiment for illustrative purposes and serves as a representative testbed; and the present invention should not be construed to be limited thereto.
An aspect of an embodiment of the present invention may include pre-clinical therapies for VML repair, with an emphasis on those which utilize dECM, and addresses the need for advanced biomanufacturing enabled by bioprinting. In this context, an aspect of an embodiment of the present invention provides, among other things, a non-classical bioprinting method, system, and a focus of applying it to a representative skeletal muscle repair technology. Also provided herein are preliminary data that highlight the manufacturing challenges addressed by this subset of bioprinting applications. Additionally, other aspects of embodiments will show, among other things, how success in this realm may be more broadly applied to other tissue engineering applications.
Here, the terms biomanufacturing and biofabrication are used interchangeably, and both refer to the process of creating a biological product, including but not limited to the use of bioprinting and bioassembly-type technologies to structure cells and materials40. More specifically, creation of affordable and scalable tissue engineered products will require simultaneously reducing production time and manufacturing costs while enabling scaling. In this regard, bioprinting can not only be used to produce complex, three dimensional structures, but also as a technology that facilitates the automated manufacturing of cell-dense constructs41-44 in a manner that can meet the regulatory requirements of a biomanufacturing process.
An aspect of an embodiment of the present invention shall provide, among other things, a critical role, which bioprinting shall play in the tissue engineering/regenerative medicine space. Specifically, for example, an aspect of an embodiment of the present invention provides, among other things, a technique, method, and system that utilizes bioprinting and sheet-based biofabrication processes. This hybrid biofabrication method is conceptually depicted in
TEMR provides a particularly relevant technology for considering the specific challenges, progress, and biomanufacturing potential of bioprinting, as an Investigational New Drug (IND) application that has been submitted by the present inventor to the FDA for the use of this technology in a pilot clinical study for secondary revision of cleft lip. Even at such an early stage in the clinical development cycle of this technology, it is worth considering how advanced biomanufacturing methods could impact clinical translation. In that regard, the novel biofabrication system and method discussed herein has the potential to provide a platform not only for development of implantable skeletal muscle repair technologies, but for a range of additional clinical applications as well—as will be discussed in more detail herein.
An aspect of an embodiment of the present invention bioprinting provides, among other things, a vast potential to enhance the development, manufacturing and scalability of tissue engineering and regenerative medicine technologies for a variety of research and clinical applications. The possibilities range in sophistication from the creation of the complex 3D tissue architectures required for biofabrication of solid organs, to the production of organoids for in vitro investigations. An aspect of an embodiment includes various roles for the use of bioprinting. For example, an aspect of an embodiment includes utilizing bioprinting to automate biomanufacturing of simpler tissue structures, such as the uniform deposition of (mono) layers of progenitor cells on sheet-like decellularized extracellular matrices (dECM). In this scenario, dECM provides a biodegradable cell-delivery matrix for creation of enhanced regenerative microenvironments following in vivo implantation. In fact, as discussed above, previous work by the present inventor has demonstrated that inclusion of muscle progenitor cells on a porcine bladder acellular matrix (BAM) for treatment of rodent volumetric muscle loss (VML) injuries significantly improved tissue repair, volume reconstitution, and functional outcomes. The present inventor refers to this implantable technology platform as tissue engineered muscle repair (TEMR). An aspect of an embodiment of the present invention provides for, among other things, bioprinting the automated fabrication of TEMR constructs with high reproducibility and scalability, while reducing costs and production timelines. The present inventor submits that such bioprinting applications are a critical component to the further enrichment of the overall biomanufacturing paradigm. In particular, for biofabrication of sheet-like implantable constructs with cells deposited on both sides—a process that is both scaffold and cell type agnostic, and furthermore, is amenable to many additional tissue engineering applications beyond skeletal muscle.
Moreover, it should be appreciated that any of the components or modules referred to with regards to any of the present invention embodiments discussed herein, may be integrally or separately formed with one another. Further, redundant functions or structures of the components or modules may be implemented. Moreover, the various components may be communicated locally and/or remotely with any user or machine/system/computer/processor. Moreover, the various components may be in communication via wireless and/or hardwire or other desirable and available communication means, systems and hardware. Moreover, various components and modules may be substituted with other modules or components that provide similar functions.
It should be appreciated that the device and related components discussed herein may take on all shapes along the entire continual geometric spectrum of manipulation of x, y and z planes to provide and meet the environmental, anatomical, and structural demands and operational requirements. Moreover, locations and alignments of the various components may vary as desired or required.
It should be appreciated that various sizes, dimensions, contours, rigidity, shapes, flexibility and materials of any of the components or portions of components in the various embodiments discussed throughout may be varied and utilized as desired or required.
It should be appreciated that while some dimensions are provided on the aforementioned figures, the device may constitute various sizes, dimensions, contours, rigidity, shapes, flexibility and materials as it pertains to the components or portions of components of the device, and therefore may be varied and utilized as desired or required.
Although example embodiments of the present disclosure are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the present disclosure be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.
It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and/or to the other particular value.
By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.
In describing example embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to accomplish a similar purpose. It is also to be understood that the mention of one or more steps of a method does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Steps of a method may be performed in a different order than those described herein without departing from the scope of the present disclosure. Similarly, it is also to be understood that the mention of one or more components in a device or system does not preclude the presence of additional components or intervening components between those components expressly identified.
Some references, which may include various patents, patent applications, and publications, are cited in a reference list and discussed in the disclosure provided herein. The citation and/or discussion of such references is provided merely to clarify the description of the present disclosure and is not an admission that any such reference is “prior art” to any aspects of the present disclosure described herein. In terms of notation, “[n]” corresponds to the nth reference in the list. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.
It should be appreciated that as discussed herein, a subject may be a human or any animal. It should be appreciated that an animal may be a variety of any applicable type, including, but not limited thereto, mammal, veterinarian animal, livestock animal or pet type animal, etc. As an example, the animal may be a laboratory animal specifically selected to have certain characteristics similar to human (e.g. rat, dog, pig, monkey), etc. It should be appreciated that the subject may be any applicable human patient, for example.
As discussed herein, a “subject” may be any applicable human, animal, or other organism, living or dead, or other biological or molecular structure or chemical environment, and may relate to particular components of the subject, for instance specific tissues or fluids of a subject (e.g., human tissue in a particular area of the body of a living subject), which may be in a particular location of the subject, referred to herein as an “area of interest” or a “region of interest.”
The term “about,” as used herein, means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. In one aspect, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, 4.24, and 5). Similarly, numerical ranges recited herein by endpoints include subranges subsumed within that range (e.g. 1 to 5 includes 1-1.5, 1.5-2, 2-2.75, 2.75-3, 3-3.90, 3.90-4, 4-4.24, 4.24-5, 2-5, 3-5, 1-4, and 2-4). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.”
The aspects of embodiments of the invention itself, together with further objects and attendant advantages, will best be understood by reference to the following detailed description, taken in conjunction with the accompanying drawings.
These and other objects, along with advantages and features of various aspects of embodiments of the invention disclosed herein, will be made more apparent from the description, drawings and claims that follow.
The foregoing and other objects, features and advantages of the present invention, as well as the invention itself, will be more fully understood from the following description of preferred embodiments, when read together with the accompanying drawings.
The accompanying drawings, which are incorporated into and form a part of the instant specification, illustrate several aspects and embodiments of the present invention and, together with the description herein, serve to explain the principles of the invention. The drawings are provided only for the purpose of illustrating select embodiments of the invention and are not to be construed as limiting the invention.
As shown substantially in the bottom portion of
After deciding to use HA as the bioink in this system, printability of HA gel was assessed. Several different weight percentages of HA ranging from 0.5% to 3% were qualitatively assessed (data not shown) and 2% HA by weight was determined to be the optimal formulation for the purposes of this project due to reasonable shape retention, ease of syringe loading, and reliable deposition. It should be appreciated that other levels of percent HA by weight may be implemented as desired or required.
The BAM scaffold for the bioprinted TEMR can be prepared in the same manner as an aspect of an embodiment of the present invention TEMR manufacturing methods described above, and in previously published work11. Both the cell-rich bioink, and the ECM-derived BAM substrate onto which the cells are deposited, play a supportive role during the maturation of a layer of tissue. In this scenario, the bioink serves only to control uniform high-density cell deposition across the entire area of the dECM scaffold (
By traditional manual methods, the process requires a total time of 15-17 days. Referring to
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An aspect of an embodiment of the present invention bioprinting method and system have overcome a broad number of manufacturing challenges. An aspect of an embodiment of the present invention bioprinting method and system provide, but not limited thereto, the following characteristics and advantages s: 1) reproducible deposition of cells/material, 2) automation and reduction of labor, 3) reduction of manufacturing cost/time, 4) method compatibility across cell types, and 5) development of a closed-loop system.
An aspect of an embodiment of the present invention next-generation bioprinted TEMR biofabrication process from bioink formulation to bioreactor preconditioning include a variety of steps and activities, some of which may include, but not limited thereto, the following: 1) choosing a bioink material and developing methods to combine cells homogenously throughout the gel while maintaining viability, 2) developing methods to load the syringe with minimal shear force and introduction of air bubbles, 3) developing a holder to drape the BAM taut and provide a relatively flat surface for printing, 4) developing a reliable method for zeroing the printhead on the ECM scaffold—reducing shear to preserve cell viability, while ensuring an even, precise print, and 5) ensuring that the system allows for bioreactor preconditioning of the cells on the scaffold with future possibility of automation.
In preparation for an aspect of an embodiment of the present invention bioprinting, the BAM scaffold 1, or other type of scaffold as desired or required, is draped over the bioassembly device 13 (See
In continuation of an aspect of an embodiment of the present invention TEMR biomanufacturing process, the seeded BAMs are transferred to differentiation media in the aforementioned cyclic stretch bioreactor 41 after another 24 hrs (see
Practice of an aspect of an embodiment (or embodiments) of the invention will be still more fully understood from the following examples and experimental results, which are presented herein for illustration only and should not be construed as limiting the invention in any way.
In order to assess the reproducibility and heterogeneity of the cell-laden bioink, immortalized mouse myoblasts (C2C12s) were printed onto glass slides. The 2% HA bioink was prepared with C2C12s as described above, and eight rectangular constructs (21 mm×16 mm×0.5 mm) were printed consecutively. Each print consisted of 138 μL of gel, resulting in a total of more than 1.1 mL of gel deposited. After 24 hours in culture, cells were stained using ReadyProbes® for F-actin and DAPI. Confocal microscopy with a 10× objective was used to perform a tile scan of the entire 21 mm×16 mm printed area for each print.
Another set of experiments explored the compatibility of these bioprinting methods with various cell types relevant to skeletal muscle tissue engineering. This included human skeletal muscle progenitor cells, human neurons, mouse endothelial cells, and C2C12s (immortalized mouse myoblasts; see cell sources below). Briefly, each of these cell types were combined into 2% HA gel and printed onto the BAM scaffold either individually, or in co-culture as further described below. Human muscle progenitor cells (hMPCs) were printed alone in 2% HA, then stained for DAPI and F-actin after 24 hours. The hMPCs were also printed in combination with human neurons. For this co-culture, the hMPCs were printed, then the human neurons were printed after 24 hours. These samples were stained for β III tubulin, desmin, and DAPI, and imaged after 13 days. The C2C12s were printed alone and stained for F-actin and DAPI after 24 hours. Finally, the C2C12s were also printed with endothelial cells by combining both cell types into a single bioink. These co-culture samples were stained for CD31, desmin, and DAPI, and imaged 4 days after printing.
Human skeletal muscle progenitor cells were obtained by isolation from discarded human samples, using a 2% collagenase digestion, according to established methods. Human neurons were derived from human induced pluripotent stem cells (hiPSCs). The hiPSCs were provided by the University of Virginia Stem Cell Core and differentiated into neurons. The endothelial cells used in these studies were mouse primary bladder endothelial cells obtained from CellBiologics (Chicago, Ill.).
Overcoming Technical Challenges of Bioprinting on dECM Sheets
There were at least three key technical challenges of printing on sheet-based scaffolds that would not only enable creation of bioprinted TEMR, but also enable bioprinting on sheet-based scaffolds for diverse research and clinical applications (see
Reproducible, Quantifiable Deposition Achieved with Bioprinting Approach
There are two prominent types of extrusion-based bioprinters—printers with pneumatically-driven extrusion and printers with piston-based extrusion, as shown in
The Organovo printer 31 utilizes piston-driven extrusion printing method, where the plunger 37 of the Hamilton syringe 35 is mechanically depressed in controlled, discrete increments. The rate of extrusion is a programmed parameter, which allows for consistent volumes of deposition every print, regardless of gel viscosity. The volume of gel deposited is measured using the graduations present on the syringes 35. The ability to print discrete, consistent volumes allows for deposition of a specific number of cells. Conversely, the commercially available pneumatically driven printers 31 have advantages that include the ability to print complex CAD files. As previously mentioned, the unique design of an aspect of an embodiment of the present invention bioassembly device 13 and plate 51 allows for interoperability between different types of commercially available bioprinters 31 (shown in
Determining the homogeneity of cell distribution throughout the bioink and the reproducibility of cell homogeneity from construct to construct is an important aspect of an embodiment of the present invention for establishing quality control metrics for the TEMR manufacturing process. Towards this end, homogeneity among eight consecutive prints was assessed and the resulting composite image of a representative print is shown in the micrographic depiction in
In this scenario, quantification of the surface area covered by cells was used as an approximation of the relative homogeneity of the prints—both within a single print and across print replicates. Surface coverage of cells on the slides was quantified for 10 images (pre-composite) in four randomly selected representative prints: print #2, 4, 6, and 8 (
The following results from initial proof of concept studies (using C2C12s) demonstrate the feasibility of using bioprinting to: 1) reduce the number of required cells (and thus reduced media and supplies cost); and 2) increase the homogeneity and reproducibility of cell coverage on both sides of the scaffold. Specifically, the current method of manual seeding utilizes 5.4×106 cells per side (1×106 cells/cm2), in large part, to compensate for inefficiencies in the seeding process. Whereas, an aspect of an embodiment of the present invention provides for the bioprinting cells to be encapsulated in a gel that allows for nominally better cell retention on the seeded area.
As shown in the micrographic depiction in
VML injuries result in the loss of vascular and nerve tissue, in addition to the loss of muscle. In order to develop improved biomimetic skeletal muscle constructs for both in vitro and in vivo applications, multiple cell types, including neurons, endothelial cells, vascular smooth muscle cells, and pericytes must eventually be included. As such, another key feature of an aspect of an embodiment of the present invention bioink and bioprinting system (and related method) is its compatibility with multiple relevant cell types. Thus far, the present inventor has successfully bioprinted human skeletal muscle progenitor cells (hMPCs), human induced pluripotent stem cell (hiPSC)-derived neurons, mouse myoblasts, and mouse endothelial cells (human skeletal muscle progenitor cells (hMPCs), human induced pluripotent stem cell (hiPSC)-derived neurons, mouse myoblasts, and mouse endothelial cells (ECs)) onto the BAM scaffold, using the aforementioned 2% HA bioink.
As discussed above,
Bioprinting with High (>90%) Cell Viability
The viability of several additional cell types was initially assessed for the Organovo NovoGen 3D bioprinter. The printer settings used were a lateral speed of 5 mm/s, an extrusion rate of 25-50 μm/s, and a z displacement of 250-500 μm between the printing surface and the needle of the Hamilton syringe 24 hours after extrusion. As shown in
Referring to
Overall, the various aspects of embodiments of the TEMR process described herein presents potentially important solutions to several biomanufacturing challenges such as automation, reproducibility, and time and cost reduction (cost of goods; COGs). These advantages are highlighted in Table 1. Although further rigorous investigations with more clinically relevant cells (e.g., human myoblasts) are required for confirmation of these findings with C2C12 cells, these initial observations demonstrate the presence of a reproducible and established cell monolayer 24 hours following bioprinting. The implication is that minimally, an aspect of an embodiment of the present invention shall provide for the ability to create uniform and homogeneous cell populations on both sides of the scaffold with a ≈7-fold reduction in the number of cells required. This may also reduce the manufacturing time line prior to bioreactor preconditioning—in effect resulting in a potential 30-85% reduction in the overall timeline for TEMR production.
As previously mentioned, an aspect of an embodiment of the present invention provides for the bioassembly device and printing plate that lends itself to, among other things, a more automated, and eventually, closed-loop system. This early stage proof of concept work lays the basis for the further development of a fully-automated, closed loop system from cell seeding to TEMR construct completion. This would be a system in which the cells could be printed on the BAMs, and then cultured, differentiated, and preconditioned all within the same bioreactor device. This approach would further reduce the manual labor required for biofabrication of TEMR, and thus, accordingly reduce the cost associated with production. When considering the biofabrication process for TEMRs in a good manufacturing practices (GMP) facility, a fully-automated, closed-loop system would also be beneficial for maintaining sterility of the product and minimizing contamination.
The biomanufacturing methods described are somewhat analogous to cell sheet technologies—another area of biofabrication research that yields cell-dense constructs. However, an aspect of an embodiment the present invention manufacturing methods for bioprinting TEMR differ from cell sheets in that an aspect of an embodiment of the bioprinting offers controllable deposition of both cell types and cell numbers, and the supporting dECM substrate itself plays a critical role in the construct. This robust, but ultimately biodegradable dECM allows force transduction to the differentiating muscle progenitor cells, facilitating cellular organization and unidirectional orientation during cyclic mechanical stretch preconditioning in the bioreactor. The dECM material is also suturable, and thus ideal for surgical implantation, ultimately enabling an improved interface with surrounding native tissue. Eventually, the dECM scaffold will degrade, leaving only remodelled/repaired/regenerated tissue structure(s) behind.
This hybrid approach of using bioprinting to establish cell sheets supported by a degradable substrate thus leverages strengths of both computer-directed printing and self-assembly (for example, see
Still referring to an aspect of an embodiment of the present invention, beyond the technical advantages that should result in accelerated biomanufacturing and reduced costs, the sheet-based platform has many potential application advantages as well (for example, but not limited thereto, see Table 2). Overall, there is considerable flexibility in a sheet-based tissue engineering platform to produce implantable constructs with very distinct geometries. Specifically, the rationale for the initial application of an embodiment of the present invention construct for craniofacial reconstruction, is related to the sheet-like nature of many of the facial muscles, for example, the orbicularis oris muscle of the lip that is the locus of cleft lip deformities. In addition, an aspect of an embodiment of the present invention system is able to leverage the double-sided printing capabilities, which has important implications for extending the range of applications. For example, tissues such as blood vessels and gastrointestinal tract could be created by printing endothelial or epithelial cells, respectively, on one side of the scaffold and smooth muscle cells on the other—followed by rolling the construct into a tubular shape. Various bioengineered constructs could leverage an aspect of an embodiment of the present invention bioprinting system described herein, and yet serve to provide tissue constructs for distinct replacement/reconstruction purposes.
In addition, an aspect of an embodiment of these sheet-like constructs of the present invention can be folded in unique ways to produce a sac-like (bag) structure that might be amenable, for example, to bladder reconstruction. There is also no obvious constraint on the size of the constructs that can be seeded, so there is opportunity for significant scalability to meet the needs of larger reconstructive procedures. In an embodiment, the constructs could also be stacked in vivo, over time, to produce even larger volumes of tissue reconstitution. This is consistent with the present inventor's published11-13 and unpublished data where implantation of TEMR constructs that range from ≈500 μm to −1 mm in thickness results in robust volume reconstitution of several millimeters in tissue thickness. While engineered constructs are often limited by the diffusion distance of oxygen, TEMR has been shown to have therapeutic effects after implantation without the presence of mature vasculature—as documented by the preclinical success of TEMR implantation8-15,26. This is presumably related to the fact that following TEMR implantation, vasculature is able to infiltrate the construct without requiring a mature vasculature in the construct itself, at the time of implantation. Finally, an aspect of an embodiment of the present invention multiple cell types can eventually be added (bioprinted) to the constructs with high spatial resolution, which when combined with additional bioreactor incubation and conditioning protocols, can produce more mature constructs, with diverse applications for biological assays (in vitro) and clinical implants (in vivo). Again, all of these advantages are summarized, at least in part, in Table 2.
Overall, an aspect of an embodiment of the present invention provides a bioprinting approach, method and system that, among other things, employs bioprinting in a non-classical method, which allows for printing high densities of cells onto sheet-like scaffolds. An aspect of an embodiment of the present invention provides an important step forward with respect to addressing very important technology gaps for the field. Moreover, an aspect of an embodiment of the present invention system and method have the potential to significantly reduce biofabrication time lines and manufacturing costs, while maintaining an open design architecture to ensure a seamless transition for any future biomanufacturing requirements. The preliminary results discussed and disclosed herein document the initial feasibility of using an aspect of an embodiment of the present invention bioprinting methods and systems to reduce the time and cost associated with biofabricating tissue engineered constructs.
In the current instance, the TEMR construct was highlighted as an example of the potential utility of this technology. Using an aspect of an embodiment of the present invention provides for bioprinting as part of the TEMR biofabrication process should enable creation of more uniform and homogeneous cell populations on both sides of the scaffold with a ≈7-fold reduction in the number of cells required. This may eventually also reduce the manufacturing time line prior to bioreactor preconditioning by as much as 90%. Certainly, further characterization and optimization may be required and is considered part of the present invention, and may be employed within the context of the invention. Nonetheless, the increased efficiencies, diminished production timelines and costs, and the wide range of potential clinical applications bode well for the utility of an aspect of an embodiment of the present invention approach, method and system as an attractive biomanufacturing platform—with promise for accelerating the application of tissue engineering/regenerative medicine technologies for diverse unmet clinical needs.
An aspect of an embodiment of the present invention provides, among other things, a bioprinting method, wherein the method may comprise: disposing a scaffold onto a bioassembly device; disposing the bioassembly device, with the scaffold, onto a bioprinter; bioprinting onto a first side of the scaffold or both the first side and a second side of the scaffold, which is disposed on the bioassembly device that is disposed on the bioprinter; transferring the bioprinted scaffold, which is disposed on the bioassembly device, onto a bioreactor; and creating tissue engineered construct while the bioprinted scaffold remains on the bioassembly device and in the bioreactor.
The method of example 1, wherein the scaffold comprises a sheet-based scaffold.
The method of example 1 (as well as subject matter in whole or in part of example 2), wherein the tissue engineered construct comprises at least one or more of any combination of the following:
implantable tissue engineered construct;
three dimensional structure tissue engineered construct;
solid organs construct;
organoids construct;
sheet-like construct;
varying geometrical shapes of the construct; and
distinct consistency on a first side of the contrast relative to a second side of the construct.
The method of example 3 (as well as subject matter in whole or in part of example 2), further comprising:
folding the sheet-like construct.
The method of example 3 (as well as subject matter of one or more of any combination of examples 2 or 4, in whole or in part), further comprising:
repeating steps of example 1 one or more times, and stacking two or more of the constructs.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-5, in whole or in part), wherein the bioprinting includes directly depositing cells onto the first side of the scaffold or both the first side and a second side of the scaffold.
The method of example 6 (as well as subject matter of one or more of any combination of examples 2-5, in whole or in part), wherein the bioprinting comprises encapsulating the cells being depositing in a gel.
The method of example 6 (as well as subject matter of one or more of any combination of examples 2-5 and 7, in whole or in part), wherein the bioprinting comprises controlling the number of cells being deposited and/or type of cells being deposited.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-8, in whole or in part), wherein the bioprinting includes extruding bioink onto the first side of the scaffold or both the first side and a second side of the scaffold.
The method of example 9 (as well as subject matter of one or more of any combination of examples 2-8, in whole or in part), wherein the bioink comprises at least one or more of any combination of the following: hyaluronic acid (HA), gelatin, alginate, fibrinogen, collagen, and other biopolymers.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-10, in whole or in part), wherein the creating comprises: culturing, differentiating, and preconditioning the scaffold in the bioreactor while the scaffold remains on the bioassembly device.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-11, in whole or in part), wherein the creating comprises:
incubating the bioprinted scaffold.
The method of example 11 (as well as subject matter of one or more of any combination of examples 2-10 and 12, in whole or in part), wherein the creating comprises:
stretching the bioprinted scaffold.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-13, in whole or in part), wherein the creating comprises:
seeding the first side of the bioprinted scaffold or both the first side and a second side of the bioprinted scaffold.
The method of example 14 (as well as subject matter of one or more of any combination of examples 2-13, in whole or in part), wherein the seeding includes controlling cell seeding density and/or cell seeding consistency.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-15, in whole or in part), wherein the disposing the scaffold onto the bioassembly device includes securing the scaffold in position for the bioprinting.
The method of example 1 (as well as subject matter of one or more of any combination of examples 2-16, in whole or in part), wherein the disposing the scaffold onto the bioassembly device includes securing the scaffold in a taut position for the bioprinting.
The method of example 17 (as well as subject matter of one or more of any combination of examples 2-16, in whole or in part), wherein disposing the bioassembly device includes securing the bioassembly device to the bioprinter.
The method of example 18 (as well as subject matter of one or more of any combination of examples 2-16, in whole or in part), wherein the securing the bioassembly device to the bioprinter comprises disposing a plate on the bioprinter configured to receive the bioassembly device.
The method of example 18 (as well as subject matter of one or more of any combination of examples 2-17 and 19, in whole or in part), wherein after transferring the bioprinted scaffold that is disposed on the bioassembly device, securing the bioassembly device to the bioreactor.
The method of example 20 (as well as subject matter of one or more of any combination of examples 2-19, in whole or in part), wherein the disposing the scaffold onto the bioassembly device includes securing the scaffold in a taut position while in the bioreactor.
Example 22 An aspect of an embodiment of the present invention provides, among other things, a bioassembly device for use with a bioprinter, wherein the device may comprise: a top portion and a bottom portion that are configured to secure a scaffold there between while the bioprinter performs bioprinting onto a first side of the scaffold or both the first side and a second side of the scaffold.
The device of example 22, wherein the top portion and the bottom portion are configured to secure the bioprinted scaffold while it is transferred to a bioreactor.
The device of example 22 (as well as subject matter in whole or in part of example 23), wherein the top portion and the bottom portion are configured to:
slidably connect together with one another; or snap-fit connect with one another one another.
The device of example 23 (as well as subject matter in whole or in part of example 24), wherein the top portion and the bottom portion are configured to secure the transferred bioprinted scaffold in the bioreactor while the scaffold is created into tissue engineered construct.
The device of example 25 (as well as subject matter of one or more of any combination of examples 23-24, in whole or in part) provided in a kit, wherein the kit includes the scaffold.
The device of example 26 (as well as subject matter of one or more of any combination of examples 23-25, in whole or in part), wherein the kit provides the scaffold as the tissue engineered construct that comprises at least one or more of any combination of the following:
implantable tissue engineered construct;
three-dimensional structure tissue engineered construct;
solid organs construct;
organoids construct;
sheet-like construct;
varying geometrical shapes of the construct; and
distinct consistency on a first side of the contrast relative to a second side of the construct.
The device of example 26 (as well as subject matter of one or more of any combination of examples 23-25 and 27, in whole or in part), wherein the kit provides the scaffold in a folded configuration construct.
The device of example 26 (as well as subject matter of one or more of any combination of examples 23-25 and 27-28, in whole or in part), wherein the kit provides two or more the scaffolds wherein the two or more the scaffolds are stacked to form the construct.
The device of example 22 (as well as subject matter of one or more of any combination of examples 23-29, in whole or in part), wherein the top portion and the bottom portion are configured to secure the scaffold there between while cells are deposited onto the first side of the scaffold or both the first side and a second side of the scaffold during the bioprinting.
The device of example 30 (as well as subject matter of one or more of any combination of examples 23-29, in whole or in part), wherein the top portion and the bottom portion are configured to secure the scaffold there between while the cells are encapsulated in a gel during bioprinting.
The device of example 22 (as well as subject matter of one or more of any combination of examples 23-31, in whole or in part), wherein the top portion and the bottom portion that are configured to secure the scaffold comprises at least one or more of the following:
a frame configured to provide the scaffold securement;
a portion of a frame configured to provide the scaffold securement;
a clamp configured to provide the scaffold securement; or
bars or elongated members arranged to provide the scaffold securement.
The device of example 22 (as well as subject matter of one or more of any combination of examples 23-32, in whole or in part), wherein the securing the scaffold while in the bioprinter includes securing the scaffold in a taut position for the bioprinting.
The device of example 22 (as well as subject matter of one or more of any combination of examples 23-33, in whole or in part), wherein the top portion and the bottom portion are configured to be secured in place at a designated location in the bioprinter.
The device of example 23 (as well as subject matter of one or more of any combination of examples 24-34, in whole or in part), wherein the top portion and bottom portion are configured to be secured in place at a designated location in the bioreactor transferred therein.
The device of example 23 (as well as subject matter of one or more of any combination of examples 24-35 in whole or in part), wherein:
the securing the scaffold while in the bioprinter includes securing the scaffold in a taut position for the bioprinting; and
the securing the scaffold while in the bioreactor includes securing the scaffold in a taut position while in the bioreactor.
The device of example 22 (as well as subject matter of one or more of any combination of examples 23-36, in whole or in part) provided in a kit, wherein the kit includes the bioprinter.
The device of example 23 (as well as subject matter of one or more of any combination of examples 23-37, in whole or in part) provided in a kit, wherein the kit includes the bioprinter and the bioreactor.
An aspect of an embodiment of the present invention provides, among other things, a bioprinting system, where the system may comprise: a designated area configured for receiving a bioassembly device, which includes a scaffold disposed in the bioassembly device; and a print head configured for bioprinting onto a first side of the scaffold or both the first side and a second side of the scaffold, while the bioassembly device is in the designated area of the bioprinting system.
The system of example 39, wherein the bioprinting includes directly depositing cells onto the first side of the scaffold or both the first side and a second side of the scaffold.
The system of example 40, wherein the bioprinting comprises encapsulating the cells being depositing in a gel.
The system of example 40 (as well as subject matter in whole or in part of example 41), wherein the bioprinting comprises controlling the number of cells being deposited and/or type of cells being deposited.
The system of example 39 (as well as subject matter of one or more of any combination of examples 40-42, in whole or in part), wherein the bioprinting includes extruding bioink onto the first side of the scaffold or both the first side and a second side of the scaffold.
The system of example 39 (as well as subject matter of one or more of any combination of examples 40-43, in whole or in part), wherein the designated area is configured to secure the bioassembly device to the bioprinting system.
The system of example 39 (as well as subject matter of one or more of any combination of examples 40-44, in whole or in part), further comprising a kit, wherein the system may be provided with a bioreactor, and wherein the bioassembly device is configured to secure the bioprinted scaffold while it is transferred to the bioreactor.
The system of example 45 (as well as subject matter of one or more of any combination of examples 40-44, in whole or in part), further comprising a kit, wherein the system may be provided with a bioreactor, and wherein the bioassembly device is configured to secure the bioprinted scaffold at a designated location in the bioreactor transferred therein.
The method of using any of the devices and systems or their components or sub-components provided in any one or more of examples 22-46, in whole or in part.
The method of manufacturing any of the devices and systems or their components or sub-components provided in any one or more of examples 22-46, in whole or in part.
A non-transitory machine readable medium including instructions for bioprinting, which when executed by a machine, causes the machine to perform any of the steps or activities provided in any one or more of examples 1-21.
A non-transitory computer readable medium including program instructions for bioprinting, wherein execution of the program instructions by one or more processors of a computer system causes the processor to carry out: any of the steps or activities provided in any one or more of examples 1-21.
The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein, and which are not admitted to be prior art with respect to the present invention by inclusion in this section.
The devices, systems, apparatuses, compositions, materials, machine readable medium, computer program products, and methods of various embodiments of the invention disclosed herein may utilize aspects (such as devices, systems, apparatuses, compositions, materials, machine readable medium, computer program products, and methods) disclosed in the following references, applications, publications and patents and which are hereby incorporated by reference herein in their entirety, and which are not admitted to be prior art with respect to the present invention by inclusion in this section:
Unless clearly specified to the contrary, there is no requirement for any particular described or illustrated activity or element, any particular sequence or such activities, any particular size, speed, material, duration, contour, dimension or frequency, or any particularly interrelationship of such elements. Moreover, any activity can be repeated, any activity can be performed by multiple entities, and/or any element can be duplicated. Further, any activity or element can be excluded, the sequence of activities can vary, and/or the interrelationship of elements can vary. It should be appreciated that aspects of the present invention may have a variety of sizes, contours, shapes, compositions and materials as desired or required.
In summary, while the present invention has been described with respect to specific embodiments, many modifications, variations, alterations, substitutions, and equivalents will be apparent to those skilled in the art. The present invention is not to be limited in scope by the specific embodiment described herein. Indeed, various modifications of the present invention, in addition to those described herein, will be apparent to those of skill in the art from the foregoing description and accompanying drawings. Accordingly, the invention is to be considered as limited only by the spirit and scope of the following claims, including all modifications and equivalents.
Still other embodiments will become readily apparent to those skilled in this art from reading the above-recited detailed description and drawings of certain exemplary embodiments. It should be understood that numerous variations, modifications, and additional embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of this application. For example, regardless of the content of any portion (e.g., title, field, background, summary, abstract, drawing figure, etc.) of this application, unless clearly specified to the contrary, there is no requirement for the inclusion in any claim herein or of any application claiming priority hereto of any particular described or illustrated activity or element, any particular sequence of such activities, or any particular interrelationship of such elements. Moreover, any activity can be repeated, any activity can be performed by multiple entities, and/or any element can be duplicated. Further, any activity or element can be excluded, the sequence of activities can vary, and/or the interrelationship of elements can vary. Unless clearly specified to the contrary, there is no requirement for any particular described or illustrated activity or element, any particular sequence or such activities, any particular size, speed, material, dimension or frequency, or any particularly interrelationship of such elements. Accordingly, the descriptions and drawings are to be regarded as illustrative in nature, and not as restrictive. Moreover, when any number or range is described herein, unless clearly stated otherwise, that number or range is approximate. When any range is described herein, unless clearly stated otherwise, that range includes all values therein and all sub ranges therein. Any information in any material (e.g., a United States/foreign patent, United States/foreign patent application, book, article, etc.) that has been incorporated by reference herein, is only incorporated by reference to the extent that no conflict exists between such information and the other statements and drawings set forth herein. In the event of such conflict, including a conflict that would render invalid any claim herein or seeking priority hereto, then any such conflicting information in such incorporated by reference material is specifically not incorporated by reference herein.
The present application claims benefit of priority under 35 U.S.C § 119 (e) from U.S. Provisional Application Ser. No. 62/741,215, filed Oct. 4, 2018, entitled “Modular Biofabrication Platform for Diverse Tissue Engineering Applications and Related Method Thereof”; the disclosure of which is hereby incorporated by reference herein in its entirety. The present application is related to International Patent Application Serial No. PCT/US2016/051948, entitled “BIOREACTOR AND RESEEDING CHAMBER SYSTEM AND RELATED METHODS THEREOF”, filed Sep. 15, 2016; Publication No. WO 2017/048961, Mar. 23, 2017; the disclosure of which is hereby incorporated by reference herein in its entirety. The present application is related to International Patent Application Serial No. PCT/US2017/045299, entitled “BIOREACTOR CONTROLLER DEVICE AND RELATED METHOD THEREOF”, filed Aug. 3, 2017; Publication No. WO 2018/027033, Feb. 8, 2018; the disclosure of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/054744 | 10/4/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62741215 | Oct 2018 | US |
