US-12623002-B2 - Fast automated approach for the derivation of acellular extracellular matrix scaffolds from tissues

US12623002B2US 12623002 B2US12623002 B2US 12623002B2US-12623002-B2

Abstract

Provided herein are systems, devices and methods to automate and optimize the decellularization process of representative tissues, such as soft tissues, for extracellular matrix (ECM)-based scaffold and biomaterial production. The automated decellularization processes and devices significantly reduce the exposure time to reagents, minimize lot-to-lot variability, and largely preserve the native composition of the ECM from the decellularized tissue or species.

Inventors

Donald O. Freytes
Camilo A. Mora-Navarro
Andreea Badileanu

Assignees

NORTH CAROLINA STATE UNIVERSITY

Dates

Publication Date: 20260512
Application Date: 20210514

Claims (20)

1 . An automated tissue decellularization system, the system comprising: a bioreactor; a filtration device, optionally a lateral inline filtration device; a stirring mechanism; a temperature-controlled system for temperature adjustment and control of the bioreactor, and a fluid control system comprising a dosing system and/or one or more dosing pumps configured to automatically supply one or more reagents to the bioreactor, and/or to apply a feedback for in situ solubilization of ECM for downstream scaffold production, and/or remove waste from the bioreactor, wherein the automated decellularization bioreactor system is configured to decellularize a tissue, optionally a soft tissue, to produce an extracellular matrix (ECM) scaffold and/or to produce solubilized downstream ECM-products.
2 . The automated decellularization bioreactor system of claim 1 , wherein the bioreactor comprises a series of bioreactors or a plurality of bioreactors.
3 . The automated decellularization bioreactor system of claim 1 , wherein the filtration device comprises a lateral inline filtration device or a fritted filter.
4 . The automated decellularization bioreactor system of claim 1 , further comprising an in-line monitoring system, optionally wherein the in-line monitoring system comprises a spectroscopy-based monitoring system, further optionally wherein the spectroscopy-based monitoring system comprises fluorescence, luminescence, absorbance and/or Raman.
5 . The automated decellularization bioreactor system of claim 4 , wherein the spectroscopy-based monitoring system is configured to measure nucleic acid removal in the decellularization system, optionally wherein the monitoring is in real-time, optionally wherein the monitoring comprises measurement of an absorbance at 260 nm.
6 . The automated decellularization bioreactor system of claim 1 , further comprising a remote dosing controller configured to control the automated tissue decellularization system.
7 . Automated decellularization bioreactor system of claim 1 , further comprising a filter component configured to maintain a decellularized tissue within the filtration device while simultaneously allowing the removal of a liquid filtrated phase, optionally solubilized ECM-products or waste.
8 . The automated decellularization bioreactor system of claim 1 , further comprising a plurality of ports, where the plurality of ports are configured for sampling, effluent removal and/or waste removal, optionally wherein the plurality of ports are configured as a sample port for continuous sampling/monitoring process.
9 . The automated decellularization bioreactor system of claim 1 , further comprising a waste removal port positioned on the filtration device.
10 . The automated decellularization bioreactor system of claim 1 , further comprising a pump configured to remove waste from the filtration device via the waste removal port and/or a pump configured to manage a feedback system configured for downstream ECM-solubilization and/or to produce solubilized downstream ECM-products.
11 . The automated decellularization bioreactor system of claim 1 , wherein the stirring mechanism comprises one or more magnetic stir bars and a magnetic stir plate and/or wherein the stirring mechanism comprises a rotating filtration system pre-loaded with weights and/or beads, such as sterile weights-beads, optionally, wherein the stirring mechanism further comprises one or more impellers and/or baffles configured to increase turbulence for improved stirring and mixing.
12 . The automated decellularization bioreactor system of claim 1 , wherein the dosing system comprises one or more pumps connected in parallel or in series by one or more tubes, wherein the one or more tubes converge on a single input into the inline filtration device, optionally wherein the one or more pumps comprise one or more peristaltic pumps.
13 . The automated decellularization bioreactor system of claim 1 , wherein the one or more dosing pumps are connected to individual reagent reservoirs.
14 . The automated decellularization bioreactor system of claim 1 , wherein the reagents are selected from the group comprising enzymatic solutions, detergents, alcohols for delipidation, acids or bases, and/or combinations thereof.
15 . The automated decellularization bioreactor system of claim 1 , further comprising a manifold configured to arrange the one or more tubes from the one or more dosing pumps to prevent unwanted interactions between reagents.
16 . The automated decellularization bioreactor system of claim 1 , wherein the system is configured to provide adjustability of an exposure time, type of reagent, and/or order in which the tissue is exposed to a reagent.
17 . A method of producing extracellular matrix (ECM) based biomaterials or ECM scaffolds, the method comprising: providing an automated decellularization bioreactor system of claim 1 ; performing a size-reducing pre-treating step of a tissue, optionally a soft tissue, to increase surface area of the tissue; and processing the pre-treated tissue through the automated decellularization bioreactor system.
18 . The method of claim 17 , wherein the size-reducing pre-treating step comprises grinding, mincing, chopping and/or micronization of the tissue.
19 . The method of claim 17 , wherein the soft tissue comprises heart tissue, vocal fold lamina propria, lung tissue, skeletal muscle tissue, pancreatic tissue, oral mucosa, supraglottic and dermis tissue, optionally wherein the tissue is provided from different source donors, further optionally wherein the source donors are human, murine, porcine or bovine source donors.
20 . The method of claim 17 , further comprising a series of hypotonic and/or hypertonic short washes and mechanical dissociation of the tissue via stirring.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Phase Application of PCT International Patent Application No. PCT/US2021/032593, filed May 14, 2021, incorporated herein by reference in its entirety, and which claims benefit of U.S. Provisional Patent Application Ser. No. 63/024,870, filed May 14, 2020, the disclosure of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under grant numbers DC017139 and DC017743 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD Provided herein are fast automated approaches for the derivation of acellular extracellular matrix scaffolds from tissues. Devices, systems and methods for deriving acellular extracellular matrix scaffolds from tissues are provided. BACKGROUND Limited availability of donor tissues and/or organs for allotransplantation combined with an increase in donor site morbidity risk associated with autografting, have led to a high demand for off-the-shelf tissue replacements.1 Extracellular matrix (ECM) scaffolds are complex, three-dimensional, heterogeneous networks of structural and functional proteins (e.g., collagens, elastins, fibronectins, laminins, etc.) that promote tissue-specific remodeling, repair, and regeneration at the site of implantation.2-3 ECM-based biomaterials are commonly derived from decellularized organs or tissues from various species (e.g., human, porcine, bovine, murine, etc.) in a process that aims at removing immunogenic cellular content while maintaining the integrity of the ECM.4 The ultimate goal is to produce a bioactive ECM-material that promotes tissue remodeling while lacking immunogenicity and eliminating any adverse host immune reactions.5-6 ECM scaffolds can be derived from a variety of organs and tissues such as, but not limited to, hearts, vocal folds (vocal cords), lungs, skeletal muscle, pancreas, and dermis with different composition and structure.4, 7 Even though, the decellularization process is not limited to a particular specie mainly because it can be adjusted depending on the source, porcine is a common model used to develop decellularization protocols. Porcine tissues are readily available given their ubiquitous use for human consumption and represent an abundant source of tissue for the derivation of ECM biomaterials such as, but not limited to, hydrogels.8 ECM hydrogels can be used both for in vitro cell culture platforms as well as in vivo therapies (e.g., coatings, hybrids, embedded with cells and/or growth factors etc.). ECM scaffolds are currently used for variety of applications such as implantable or injectable materials, ECM-based bio-inks, and substrates for cell growth.8-9 Depending on the application, ECM scaffolds can be processed into various formulations such as single sheets, multi-laminated sheets, powders, and hydrogels.10 Many of these materials are either in preclinical stages or have become commercially available.11 Patches or sheets derived from skin, small intestine submucosa (SIS), and urinary bladder matrix (UBM) are commonly used for various clinical applications that include, but are not limited to, burn wounds, diabetic ulcers, etc. However, ECM sheets require surgical access when implanted at the wound site and cannot be delivered via minimally invasive techniques.12 Current decellularization protocols use prolonged exposure times to each chemical or biological washes that could result in potential disruption of important ECM components leading to a decrease in the inherent bioactivity.13 Chemical agents (e.g. acids and bases, non-ionic and ionic detergents etc.) and biological agents (e.g. enzymes, chelating agents, etc.) are known to disrupt the ECM ultrastructure and damage important ECM components such as collagens, glycosaminoglycans (GAGs), elastins, and laminin if utilized for extended periods of time.14 For example, a commonly used decellularization reagent, sodium dodecyl sulfate (SDS), can effectively remove immunogenic cellular and nuclear content (e.g., DNA). However, SDS can affect the overall ultrastructure of the ECM and damage collagens, GAGs, and growth factors.4 Another example includes sodium deoxycholate, which can disrupt the ECM ultrastructure and remove GAGs.15 Decellularization reagents are typically put in contact with tissue sheets and subjected to agitation and manual liquid changes. This decellularization approach can result in nonconformities in the homogeneity of the ECM derived-product due to the inherent variability of a manual decellularization process. Currently, there is no standard decellularization protocol available and each protocol has to be optimized for the specific tissue source, specie, and configuration (sheet vs. whole organ decellularization).9 Depending on pre- and post-decellularization processing steps, manual-labor, biofabrication method, tissue source, size