Patent Application
20220372446
Provided herein are floating hydrogel droplet culture methods that enable scaling of stem cell derived alveolar epithelial cell (AEC) expansion to numbers compatible with large animal or human whole lung engineering, as well as molds for generating the droplets and methods of use thereof.
Induced pluripotent stem cell derived alveolar epithelial cells (iPSC-AECs) are a patient-specific cell source for bio-engineering of human pulmonary epithelium. Disease modeling and therapeutic applications require cost effective and technically feasible differentiation and expansion protocols.
Provided herein are floating hydrogel droplet culture methods that enable scaling of stem cell derived alveolar epithelial cell (AEC) expansion to numbers compatible with large animal or human whole lung engineering. Stable cellular phenotype was documented through both culture expansion and biomimetic lung culture. These methods can be used for human scale whole organ lung generation.
Thus provided herein are methods for generating an expanded population of alveolar epithelial cells (AECs). The methods include (a) providing a first population of AECs; (b) mixing the first population of AECs into a hydrogel precursor; (c) allowing or promoting gelation of the hydrogel precursor to form a droplet; and (d) culturing the droplets in suspension in moving media sufficient for expansion of the first population, thereby generating an expanded population of AECs. In some embodiments, after step (b), the methods include transferring the mixture to a mold apparatus as described herein, and then after gelation of the hydrogel precursor in step (c), removing the droplet from the mold apparatus.
In some embodiments, the first population of AECs comprises induced pluripotent stem cell (iPSC)-derived AECs.
In some embodiments, the iPSC-derived AECs are obtained by a method comprising: providing an initial population of iPSC; culturing the iPSC under conditions sufficient for definitive endodermal differentiation, then under conditions sufficient for anteriorized endodermal differentiation, and then under conditions sufficient for ventralized endodermal differentiation, thereby obtaining a population of iPSC-derived AECs.
In some embodiments, the droplet has a maximal diameter of 2-10 mm.
In some embodiments, the hydrogel is a natural or synthetic hydrogel scaffold. In some embodiments, the natural hydrogel scaffold comprises extracellular matrix (ECM), collagen, fibrin, bone sialoprotein, vitronectin, alginate, or laminin. In some embodiments, the synthetic hydrogel scaffold comprises a synthetic polymeric scaffold selected from poly(2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide) (PMEDSAH), polyacrylamide (PAM), poly(sodium 4-stryenesulfonate) (PSS), poly(methyl vinylether-alt-maleic anhydride), and poly(ethylene glycol) (PEG) hydrogels.
In some embodiments, allowing or promoting gelation of the hydrogel comprises providing a temperature, chemical, or light sufficient to initiate crosslinking of the hydrogel scaffold.
In some embodiments, the moving media is spinning or flowing culture.
In some embodiments, the expanded population of AECs comprises cells that express Nkx2.1 and aquaporin 5 (AQP5) or Surfactant Protein C (SPC).
Also provided herein are expanded populations of AECs produced by a method described herein.
Additionally, provided herein are methods for providing a bioartificial lung organ. The methods include oroviding an expanded population of AECs produced by a method described herein; providing a (cell-free) lung tissue matrix (e.g., from a human or pig) including an airway and vasculature; seeding the lung tissue matrix with the expanded population of AECs through the airway, with endothelial cells through the vasculature, and with mesenchymal cells through either one or both of the airway and the vasculature; and maintaining the matrix under conditions sufficient for the formation of a functional epithelium in the airways and functional vasculature. Also provided herein are bioartificial lung organs produced by a method described herein.
Further, provided herein is a mold apparatus, comprising: a mold body comprising a polymeric material, the mold body defining a first cavity and a second cavity, the first and second cavities each having a radius of between 0.5 mm and 5 mm and configured to receive a composition, the mold body further defining a first channel that extends along a longitudinal axis that intersects the first and second cavities, wherein the first channel is defined by a depth dimension configured to limit a volume amount of the composition in the first and second cavities. In some embodiments, the polymeric material is flexible. Also provided is a mold apparatus comprising: a flexible body defining a plurality of cavities, the plurality of cavities forming an array pattern comprising at least first and second rows, wherein each row comprises at least two or more cavities aligned along first and second longitudinal axes, respectively, the first and second longitudinal axes being spaced apart from one another by a separation distance, wherein each cavity is configured to form semi-spherical shaped compositions, and the cavities each have a radius of between 0.5 mm and 5 mm and are configured to receive a composition, wherein the cavities are defined by a depth dimension configured to limit a volume amount of the composition in the first and second cavities.
In some embodiments, the flexible body is formed from a polymeric material
In some embodiments, the flexible material is selected from the group consisting of silicones and polyurethanes. In some embodiments, the polymeric material comprises polydimethylsiloxane (PDMS).
In some embodiments, each cavity (e.g., the bottom of each cavity) is defined by a hemispherically shaped surface. In some embodiments, each cavity is configured to form spherically shape compositions or hemi-spherically shaped compositions. In some embodiments, the first channel extends from one side edge of the mold body to a second, opposite side edge.
In some embodiments, the depth dimension is configured to limit the volume of the composition in each cavity to a maximum volume amount of about 50 μL to about 150 μL.
Also provided herein are methods of forming shaped gel compositions, the method comprising adding a composition to cavities of a mold apparatus as described herein, the composition being a liquid comprising a biologic; forming a plurality of semi-solid or solid compositions in the cavities of the mold; and removing the semi-solid or solid compositions from the cavities of the mold.
In some embodiments, the liquid is a hydrogel precursor and the biologic comprises cells.
In some embodiments, the semi-solid or solid composition is a hydrogel.
In some embodiments, the removing step comprises flexing the body of the mold.
In some embodiments, the semi-solid or solid compositions retain a predetermined shape, e.g., in a spin culture, e.g., for at least 1 day, at least 5 days, or at least 10 days.
In some embodiments, the semi-solid or solid compositions are spherical or semi-spherical.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Currently, more than 1300 patients are awaiting a life-saving lung transplant in the United States (1). Of these 1300 patients, approximately 300 patients will die awaiting a pulmonary transplant (2). The fortunate patients who receive a lung transplant still require intensive immunosuppression, which is associated with significant morbidity (3). Lung bioengineering for augmentation or replacement utilizing patient-specific cell populations have the potential to provide an alternative to donor lungs and solve both donor organ shortage and the need for immunosuppression.
Any therapy that aims to replace gas exchange tissue, be it organ engineering or delivery of a cell therapy, depend on the availability of sufficient numbers of human pulmonary epithelial cells. Billions of distal lung epithelial cells from induced pluripotent stem cells (iPSC) are needed to adequately recellularize whole organ lung constructs. This need could be met utilizing directed differentiation of iPSCs toward lung-lineage committed cells. A recently published protocol has been adapted by our laboratory to reproducibly generate type II alveolar epithelial cells (AECs) via directed differentiation and fluorescent sorting of an iPSC line which has been genetically modified to carry fluorescent lung lineage markers (4-6). The resulting cells form alveolar spheres when encapsulated in adherent 3D Matrigel droplets to yield type II AECs from human iPSCs (iPSC-AECs). Type II pneumocytes secrete surfactant which supports alveolar maintenance via reduction of aqueous surface tension and also serve as a reservoir progenitor cell population for type I pneumocytes which facilitate gas exchange (7).
Perfusion-decellularization of rat or human lungs to generate extracellular matrix (ECM) scaffolds suitable for recellularization with iPSC-AECs was previously reported (8, 9). Upscale of this concept to human lungs requires approximately 10.5 billion epithelial cells (10). The established adherent hydrogel droplet culture (4) limits current capacity for large-scale organ recellularization with respect to culture time and resources.
The methods described herein are straightforward cell culture methods for iPSC-AEC expansion that can be scaled for large animal or human lung bioengineering. Matrigel is known to support differentiation and proliferation of iPSC-AECs, but there are challenges associated with its use (8, 9, 14). Matrigel is a liquid only at cold temperatures and rapidly undergoes a gel transition at 37° C., making it difficult to handle (14). The present method speeds droplet formation while maintaining the three-dimensional droplet structure, in contrast to the previously described method in which each drop takes 90 seconds to form (4). Additionally, the floating droplet method allows for greater cell expansion. This is an improvement on previously described methods for iPSC-AEC culture with reduction in labor and physical material expenditure while increasing cell yield.
The present methods are scalable to a variety of culture sizes. The floating droplet culture vessel or volume of culture medium is easily increased or decreased for differing applications. The culture methods can be automated for large iPSC-derived cell farms for commercial applications. The phenotypic stability of the cells in this floating droplet culture system is important. An obvious concern when expanding iPSC-derived cells is transdifferentiation. Comparable metabolic activity and expression of SPC was demonstrated between cells from the two culture methods, while a significant increase in both the cell culture yield and markers of proliferation (Ki67 expression) was seen in the floating droplet culture. The predilection to spontaneously form alveolar spheroids was also preserved on histological review of the cells from the floating droplet culture. When these cells were seeded on a native ECM biomimetic lung culture, they appropriately populated the distal airways with a columnar type epithelium with comparable metabolic activity as evidenced by the resazurin assay and biochemical markers bicarbonate, lactate, and glucose.
Priming iPSC-AECs for Differentiation
There is no established protocol for reliable differentiation of iPSC-derived type I AECs. Yamamoto et al described a subpopulation of iPSC-derived type I AECs identified incidentally when describing a type II AEC differentiation protocol, but this pertained to a very small portion of the differentiated cells (15). An intriguing finding in the present study is the difference in SPC and AQP5 expression in the biomimetic lung culture tissues.
Methods for Generating Stem Cell-Derived Alveolar Cells
Provided herein are scalable methods for generation of stem cell-derived alveolar cells. The methods include culturing the cells in matigel droplets formed using a method described herein.
The present methods can be performed using a starting population of stem cells, e.g., cells from a human embryonic stem cell line (e.g., H9, H1) or embryonic stem cell-like (ESC-like) induced pluripotent stem cells (iPSCs), e.g., generated from primary cells autologous to a subject to be treated using a method described herein. Primary cells such as airway basal cells, lineage negative lung progenitor cells, club cells or type II pneumocytes can also be used.
Methods for generating iPSC are known in the art. In some embodiments, the methods for generating hiPSC can include obtaining a population of primary somatic cells from a subject, e.g., a subject who is afflicted with PD and in need of treatment for PD. Preferably the subject is a mammal, e.g., a human. In some embodiments, the somatic cells are fibroblasts. Fibroblasts can be obtained from connective tissue in the mammalian body, e.g., from the skin, e.g., skin from the eyelid, back of the ear, a scar (e.g., an abdominal cesarean scar), or the groin (see, e.g., Fernandes et al., Cytotechnology. 2016 March; 68(2): 223-228), e.g., using known biopsy methods. Other sources of somatic cells for hiPSC include hair keratinocytes (Raab et al., Stem Cells Int. 2014; 2014:768391), blood cells, or bone marrow mesenchymal stem cells (MSCs) (Streckfuss-Bömeke et al., Eur Heart J. 2013 September; 34(33):2618-29). In some embodiments, the primary cells (e.g., fibroblasts) are exposed to (cultured in the presence of) factors sufficient to induce reprogramming to iPSC. Peripheral blood-derived mononuclear cells can be isolated from patient blood samples and used to generate induced pluripotent stem cells. In other examples, induced pluripotent stem cells can be obtained by reprograming with constructs optimized for high co-expression of Oct4, Sox2, Klf4, c-MYC in conjunction with small molecule such as transforming growth factor β (SB431542), MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin). See Groß et al., Curr Mol Med. 13:765-76 (2013) and Hou et al., Science 341:651:654 (2013). Methods for generating endothelial cells from stem cells are reviewed in Reed et al., Br J Clin Pharmacol. 2013 April; 75(4):897-906. Cord blood stem cells can be isolated from fresh or frozen umbilical cord blood. Mesenchymal stem cells can be isolated from, for example, raw unpurified bone marrow or ficoll-purified bone marrow. Epithelial and endothelial cells can be isolated and collected from living or cadaveric donors, e.g., from the subject who will be receiving the bioartificial lung, according to methods known in the art. For example, epithelial cells can be obtained from a skin tissue sample (e.g., a punch biopsy), and endothelial cells can be obtained from a vascular tissue sample.
Although other protocols for programming can be used (e.g., as known in the art or described herein), in preferred embodiments the present methods can include introducing (contacting or expressing in the cell) four transcription factors, i.e., Oct4, Sox2, Klf4, and L-Myc, known colloquially as the as Yamanaka 4 factors (Y4F). See, e.g., Takahashi and Yamanaka, Cell. 2006; 126(4):663-676; Takahashi et al., Cell. 2007; 131(5):861-872; Yu et al. Science. 2007; 318(5858):1917-1920; Park et al., Nature. 2008; 451(7175):141-146. In some embodiments, the methods also include contacting or expressing in the cell one or more miRNAs, e.g., (i) at least one miR-302 cluster member and (ii) at least one miR-200 cluster member; see US 20160298089 and Song et al., J Clin Invest. 2020; 130(2):904-920.
The starting population of stem cells is differentiated to alveolar epithelial cells (AECs) via directed differentiation, e.g., as shown in
These steps are carried out using standard culture methods, e.g., in culture dishes. After ventralization, the cells can be fluorescence-activated cell sorted (FACS) for purification; for example, in cells that express a reporter protein, that reporter protein can be used (exemplified herein is the sorting of Nkx2.1-GFP positive cells).
The cells are then mixed in a natural or synthetic hydrogel scaffold, e.g., comprising natural extracellular matrix (ECM), e.g., MATRIGEL (Corning, Corning, N.Y.), GELTREX LDEV-Free Reduced Growth Factor Basement Membrane Matrix (GIBCO/ThermoFisher), or CULTREX Basement Membrane Extract (BME) (Trevigen); natural scaffolds comprising collagen (e.g., and collagen type IV), fibrin, bone sialoprotein, vitronectin (e.g., VITRONECTIN XF™ (STEMCELL Technologies), alginate, or laminin; synthetic polymeric scaffolds, e.g., comprising poly(2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide) (PMEDSAH), polyacrylamide (PAM), poly(sodium 4-stryenesulfonate) (PSS), poly(methyl vinylether-alt-maleic anhydride), or poly(ethylene glycol) (PEG) hydrogels (e.g., photo-crosslinked or enzymatically crosslinked PEG-vinyl sulfone (PEG-VS), photopolymerizable PEG thiol-ene hydrogel scaffolds with cysteine-flanked MMP-sensitive crosslinks, or MMP-degradable, RGD-functionalized PEG hydrogel scaffolds factor-Mk-mediated crosslinked peptide-functionalized PEG monomers), or combinations thereof. In some embodiments, additional factors are included such as heparin sulfate proteoglycans such as perlecan, or peptides are used to promote cell growth or adhesion to synthetic or natural scaffolds, e.g., laminin-derived peptide (YIGSR) or fibronectin-derived Arg-Gly-Asp (RGD) peptides, linear or circularized (cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGDfK)), e.g., SYNTHEMAX, a synthetic vitronectin scaffold functionalized with RGD (Corning). A number of suitable scaffolds are known in the art. See, e.g., Cruz-Aculia and Garcia, Matrix Biol. 2017 January; 57-580:324-333; Murrow et al., Development. 2017; 144:998-1007; Murphy et al., Nat Mater. 2014; 13:547-557; Nguyen et al., Nat Biomed Eng. 2017; 1: 0096; and Aisenbrey and Murphy, Nature Reviews Materials 5:539-551 (2020), and references cited therein. In some embodiments, the hydrogel scaffold composition comprises one or more growth factors, e.g., VEGF, FGF (e.g., bFGF), TGFbeta inhibitors, kir, Wnt inhibitors,
The cells are mixed into a hydrogel scaffold precursor (e.g., in liquid or semi-liquid form, i.e., sufficiently flowable to be easily transferred), and then the mixture is transferred to a droplet mold as described herein, and gelling is allowed or promoted, e.g., by initiation of crosslinking as appropriate to the selected hydrogel scaffold. The hydrogel has an elasticity and shear modulus (stiffness) sufficient to retain the shape of a formed droplet.
The droplets are three-dimensional. In some embodiments, the droplets are substantially spherical, ovoid, cylinder, cube, or cuboid. In some embodiments, the volume of the droplet is about 50-150 μL. In some embodiments, the droplets are 1-10 mm in diameter or width, e.g., 3-9 mm, 5-7 mm, or about 6 mm. In some embodiments, the droplets each comprise about 1,000-50,000 cells, e.g., about 10,000-30,000, e.g., about 20,000 cells.
After gelation the droplet is removed from the mold and placed into a suspension culture, e.g., a spinning or flowing culture, in media sufficient to support expansion of the cells. The droplets can be maintained in culture long enough to allow for proliferation (expanstion) of the cell population to desired levels.
The expanded populations of cells can be used, e.g., in transplantation protocols. The cells can be transplanted directly, or can be used to recellularize whole or partial organ lung constructs. Methods for making lung constructs are known in the art; see, e.g., US 20170326273; US 20170073645; U.S. Pat. No. 10,624,992.
For example, the cells can be used to seed a lung tissue matrix, e.g., introduced into the matrix through the airway (tracheal) line (epithelial cells). For example, a tissue matrix can be seeded with the expanded AECs in vitro at any appropriate cell density. In addition, a matrix comprising an airway and vasculature can be seeded with the AECs through the airway, with endothelial cells through the vasculature, and with mesenchymal cells through either one or both of the airway and the vasculature. For example, cell densities for seeding a matrix can be at least 1×103 cells/gram matrix. Cell densities can range between about 1×105 to about 1×1010 cells/gram matrix (e.g., at least 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 cells/gram matrix) can be used.
In some cases, a decellularized or artificial lung tissue matrix, as provided herein, can be seeded with the cell types and cell densities described above, e.g., by gravity flow or perfusion seeding. For example, a flow perfusion system can be used to seed the decellularized lung tissue matrix via the vascular system preserved in the tissue matrix (e.g., through the arterial line). In some cases, automated flow perfusion systems can be used under the appropriate conditions. Such perfusion seeding methods can improve seeding efficiencies and provide more uniform distribution of cells throughout the composition. Quantitative biochemical and image analysis techniques can be used to assess the distribution of seeded cells following either static or perfusion seeding methods.
In some cases, a tissue matrix can be impregnated or perfused with one or more growth factors to stimulate expansion of the seeded cells. For example, a tissue matrix can be impregnated or perfused with growth factors appropriate for the methods and materials provided herein, for example, vascular endothelial growth factor (VEGF), TGF-growth factors, bone morphogenetic proteins (e.g., BMP-1, BMP-4), platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), e.g., FGF-10, insulin-like growth factor (IGF), epidermal growth factor (EGF), or growth differentiation factor-5 (GDF-5). See, e.g., Desai and Cardoso, Respire. Res. 3:2 (2002). These growth factors can be encapsulated to control temporal release. Different parts of the scaffold can be enhanced with different growth factors to add spatial control of growth factor stimulation. In the present methods, the cells seeding the airway can be perfused with a notch inhibitor, e.g., a gamma secretase inhibitor.
Seeded tissue matrices can be incubated for a period of time (e.g., from several hours to about 14 days or more) post-seeding to improve adhesion and penetration of the cells in the tissue matrix. The seeded tissue matrix can be maintained under conditions in which at least some of the regenerative cells can multiply and/or differentiate within and on the acellular tissue matrix. Such conditions can include, without limitation, the appropriate temperature (35-38 degree centigrade) and/or pressure (e.g., atmospheric), electrical and/or mechanical activity (e.g., ventilation via positive or negative pressure with positive end expiratory pressure from 1-20 cmH2O, mean airway pressure from 5-50 cmH2O, and peak inspiratory pressure from 5-65 cmH2O), the appropriate gases, e.g., O2 (1-100% FiO2) and/or CO2 (0-10% FiCO2), an appropriate amount of humidity (10-100%), and sterile or near-sterile conditions. Such conditions can also include wet ventilation, wet to dry ventilation and dry ventilation. In some cases, nutritional supplements (e.g., nutrients and/or a carbon source such as glucose), exogenous hormones, or growth factors can be added to the seeded tissue matrix. In preferred embodiments, a notch inhibitor, e.g., a gamma secretase inhibitor, is added to the cells seeded through the airway (see, e.g., U.S. Pat. No. 10,624,992). Histology and cell staining can be performed to assay for seeded cell retention and propagation. Any appropriate method can be performed to assay for seeded cell differentiation. In general, the methods described herein will be performed in an airway organ bioreactor apparatus, e.g., as described herein.
Thus, the methods described herein can be used to generate a transplantable bioartificial lung tissue, e.g., for transplanting into a human subject. As described herein, a transplantable tissue will preferably retain a sufficiently intact vasculature that can be connected to the patient's vascular system.
The bioartificial lung tissues described herein can be combined with packaging material to generate articles of manufacture or kits. Components and methods for producing articles of manufacture are well known. In addition to the bioartificial tissues, an article of manufacture or kit can further can include, for example, one or more anti-adhesives, sterile water, pharmaceutical carriers, buffers, and/or other reagents for promoting the development of functional lung tissue in vitro and/or following transplantation. In addition, printed instructions describing how the composition contained therein can be used can be included in such articles of manufacture. The components in an article of manufacture or kit can be packaged in a variety of suitable containers.
The entire disclosures of all of the foregoing are hereby incorporated by reference herein.
Methods for Using Bioartificial Lungs
This document also provides methods and materials for using bioartificial lung tissues and, in some cases, promoting lung function. In some embodiments, the methods provided herein can be used to restore some lung function in patients having diseases that impair or reduce lung capacity (e.g., cystic fibrosis, COPD, emphysema, lung cancer, asthma, pulmonary hypertension, lung trauma, or other genetic or congenital lung abnormalities, e.g., bronchogenic cyst, pulmonary agenesis and hypoplasia, polyalveolar lobe, alveolocapillary dysplasia, sequestration including arteriovenous malformation (AVM) and scimitar syndrome, pulmonary lymphangiectasis, congenital lobar emphysema (CLE), and cystic adenomatoid malformation (CAM) and other lung cysts). The methods provided herein also include those wherein the subject is identified as in need of a particular stated treatment, e.g., increased lung function, or increased or improved lung capacity.
Bioartificial lung tissues (e.g., whole organs or portions thereof) can be generated according to the methods provided herein. In some embodiments, the methods comprise transplanting a bioartificial lung tissue as provided herein to a subject (e.g., a human patient) in need thereof. In some embodiments, a bioartificial lung tissue is transplanted to the site of diseased or damaged tissue. For example, bioartificial lung tissues can be transplanted into the chest cavity of a subject in place of (or in conjunction with) a non-functioning or poorly-functioning lung; methods for performing lung transplantation are known in the art, see, e.g., Boasquevisque et al., Surgical Techniques: Lung Transplant and Lung Volume Reduction, Proceedings of the American Thoracic Society 6:66-78 (2009); Camargo et al., Surgical maneuvers for the management of bronchial complications in lung transplantation, Eur J Cardiothorac Surg 2008; 34:1206-1209 (2008); Yoshida et al., “Surgical Technique of Experimental Lung Transplantation in Rabbits,” Ann Thorac Cardiovasc Surg. 11(1):7-11 (2005); Venuta et al., Evolving Techniques and Perspectives in Lung Transplantation, Transplantation Proceedings 37(6):2682-2683 (2005); Yang and Conte, Transplantation Proceedings 32(7):1521-1522 (2000); Gaissert and Patterson, Surgical Techniques of Single and Bilateral Lung Transplantation in The Transplantation and Replacement of Thoracic Organs, 2d ed. Springer Netherlands (1996).
The methods can include transplanting a bioartificial lung or portion thereof as provided herein during a surgical procedure to partially or completely remove a subject's lung and/or during a lung resection. The methods can also include harvesting a lung or a portion thereof from a live donor or cadaver and preserving or regenerating the lung in a bioreactor described herein. In some cases, the methods provided herein can be used to replace or supplement lung tissue and function in a subject, e.g., a human or animal subject.
Any appropriate method(s) can be performed to assay for lung function before or after transplantation. For example, methods can be performed to assess tissue healing, to assess functionality, and to assess cellular in-growth. In some cases, tissue portions can be collected and treated with a fixative such as, for example, neutral buffered formalin. Such tissue portions can be dehydrated, embedded in paraffin, and sectioned with a microtome for histological analysis. Sections can be stained with hematoxylin and eosin (H&E) and then mounted on glass slides for microscopic evaluation of morphology and cellularity. For example, histology and cell staining can be performed to detect seeded cell propagation. Assays can include functional evaluation of the transplanted tissue matrix or imaging techniques (e.g., computed tomography (CT), ultrasound, or magnetic resonance imaging (e.g., contrast-enhanced MRI)). Assays can further include functional tests under rest and physiologic stress (e.g., body plethysmography, lung function testing). Functionality of the matrix seeded with cells can be assayed using methods known in the art, e.g., histology, electron microscopy, and mechanical testing (e.g., of volume and compliance). Gas exchange can be measured as another functionality assay. To assay for cell proliferation, thymidine kinase activity can be measured by, for example, detecting thymidine incorporation. In some cases, blood tests can be performed to evaluate the function of the lungs based on levels of oxygen in the blood.
To facilitate functionality assays during culture, any line of the bioreactor apparatus' described herein may include sampling ports to allow for single or real-time measurements of functionality parameters (e.g., pH, glucose, lactate, Na, K, Ca, Cl, bicarb, O2, CO2, sat). Metabolites may also be used to monitor cell number and viability using colorimetric assays, and biochemical assays may be used to monitor cell maturation (e.g., measuring surfactant protein, etc.) For example, an increased concentration of surfactant can indicate that the culture lung possesses sufficient epithelial cells to withstand dry ventilation. In some cases, endothelial barrier function may be used as a marker of vascular maturity. Lungs can be perfused with different sizes of molecules (such as dextrans of defined sizes and albumin), and microbeads (increasing sizes from 0.2 to 5 um), as well as isolated red blood cells. Bronchoalveolar lavage fluid can then be sampled to assess leakage of these markers into the alveolar space. For example, 500-kDa dextran can be used in combination with a Bronchoalvelar lavage assay to determine the percentage of dextran retained within the vascular compartment. An increase in the percentage of dextran retained indicates an improvement in the barrier function because barrier function to dextran is dependent on viable and functional endothelium, while dextran will diffuse across a denuded vascular basement membrane (e.g., in an acellular lung) over time during constant perfusion. For example, a cadaveric lung may retain substantially all of the dextran within the vascular compartment while acellular lungs may retain a small percentage of the dextran (e.g., 10.0%±8.0%). Leakage of these markers into the alveolar space greater than a tolerated minimum (for example >10% of 4 um microbeads, or greater than 20% of 0.2 um microbeads) can be used to indicate that the lung is not sufficiently mature to withstand dry ventilation.
In some cases, molecular biology techniques such as RT-PCR can be used to quantify the expression of metabolic (e.g. surfactant protein, mucin-1) and differentiation markers (e.g. TTF-1, p63, surfactant protein C). Any appropriate RT-PCR protocol can be used. Briefly, total RNA can be collected by homogenizing a biological sample (e.g., tendon sample), performing a chloroform extraction, and extracting total RNA using a spin column (e.g., RNeasy® Mini spin column (QIAGEN, Valencia, Calif.)) or other nucleic acid-binding substrate. In other cases, markers associated with lung cells types and different stages of differentiation for such cell types can be detected using antibodies and standard immunoassays.
An exemplary airway organ bioreactor and methods of use thereof are described in WO 2015/138999, which is incorporated herein by reference in its entirety. Other exemplary bioreactors are described in Charest et al., Biomaterials. 2015 June; 52:79-87. doi: 10.1016/j.biomaterials.2015.02.016; Gilpin et al., Ann Thorac Surg. 2014 November; 98(5):1721-9; discussion 1729. doi: 10.1016/j.athoracsur.2014.05.080; Price et al., Tissue Eng Part A 2010; 16(8):2581-91; Petersen et al., Cell Transplant 2011; 20(7):1117-26; Bonvillain et al., J Vis Exp 2013; (82):e50825; Nichols et al., J Tissue Eng Regen Med. 2016 Jan. 12. doi: 10.1002/term.2113.
Provided herein is a mold apparatus configured for forming a plurality of shaped solid or semi-solid compositions containing a biologic (e.g., cells). For example, a mold body having multiple cavities (e.g., wells) described herein can be configured for high-throughput formation of semi-solid or solid biological compositions (e.g., gels), such as cell-laden hydrogel droplets as described herein. The body of the mold apparatus described herein can be made of a flexible material that advantageously allows the mold to be flexed, which in turn, facilitates the release of molded compositions within the cavities. In some cases, the mold apparatus described herein can be made of materials that are biocompatible to advantageously produce shaped materials without introducing components that can cause an adverse reaction in a subject. In some cases, the mold apparatus is made of materials having chemical, thermal and/or mechanical properties capable of withstanding stress or thermal-incurring processes, such as sterilization (e.g., using an autoclave process).
In some embodiments, the mold apparatus 800 is composed of one or more materials that are heat-resistant. For example, in some cases, the mold can be made of one or more materials that do not plastically deform at temperatures of about 135° C. or more (e.g., about 121° C. or more, about 127° C. or more). In some embodiments, the mold apparatus is made of one or more materials that is pressure-resistant. For example, in some cases, the mold can be made of materials that do not plastically deform when subjected to pressures of about 15 psi or more (e.g., about 10 psi or more, or about 12 psi or more). The mold can be made of materials that advantageously allow the mold to be compatible with thermal or chemical sterilization processes, such as an autoclave or other sterilization process. In some embodiments, the mold can be made of materials that are biologically and/or chemically inert.
In various embodiments, the mold provided herein can be a flexible mold; alternatively, the mold can be rigid. In some embodiments, the materials of the mold can be include one or more polymers. In some embodiments, the materials can include one or more elastomers. In some embodiments, the material is polyurethane, e.g., thermoplastic polyurethane (TPU). In some embodiments, the material is silicone or silicon-based, e.g., polydimethylsiloxane (PDMS). In some embodiments, the material is Poly(methyl methacrylate) (PMMA), Polycarbonate, Polystyrene, Poly(ethylene glycol) diacrylate (PEGDA), Cyclic Olefin Copolymer (COP), or Cyclic Olefin Polymer (COP). Where the mold is rigid, alternative methods can be used to remove the droplets from the wells, e.g., inclusion of a narrow channel running from the bottom of each well to the back or bottom of the mold, allowing for insertion of a wire, needle or plunger to push the droplets from the wells, or for introduction of air pressure to blow the gelled droplets from the wells.
Still referring to
The example mold apparatus 800 of
The dimensions of mold apparatus 800 or components thereof can be adjusted as desired. For example, in some cases, the dimensions of mold apparatus 800 can range in a millimeter or centimeter scale. For example, the longitudinal length and width of mold apparatus 800 can range from about 10 mm to about 100 mm, up to about 10-20 cm. In some cases, the length of the mold can range from about 30 mm to about 60 mm and the width can range from about 10 to about 40 mm. In some cases, the length of the mold can range from about 8 cm to about 10 cm and the width can range from about 4 cm to about 8 cm. The width of channel 813, wc, can range from about 0.1 mm to about 3 mm (e.g., from about 0.5 to about 2 mm, or from about 0.1 to about 1 mm) and the width of trench 814, wT, can range from about 0.1 mm to about 3 mm (e.g., from about 0.5 to about 1 mm, from about 1 mm to about 2 mm, or from about 0.75 to about 1.5 mm). Trenches 814 of mold apparatus 800 can spaced apart from a respective end of mold 800 by about 10 mm to about 25 mm. Each well 810 of mold apparatus 800 can have the same transverse dimension (e.g., radius). The radius, r, can be between 0.5 mm and 5 mm, e.g., between 2 mm and 4 mm (e.g., between 2.5 mm and 4 mm, between 3 mm and 4 mm, between 3.5 mm and 4 mm, between 2 mm and 3.5 mm, between 2 mm and 3 mm, or between 2 mm and 2.5 mm). The central axis of a well can be spaced apart from a central axis of an adjacent well (e.g., center to center distance, a separation distance, or a pitch) by about 5 to about 20 mm. In some embodiments, each well 810 of mold apparatus 800 can have a depth that is roughly equivalent to the diameter of the well 810 (e.g., 2×r), e.g., about 0.5-5 mm, optionally plus the depth of the channel 813.
Still referring to
The mold apparatus described herein can optionally define additional apertures configured to receive pipettes. For example, as shown in
The molds described herein can be used to form semi-solid or solid shaped compositions using the following steps. The wells 910 of the mold apparatus 901 (see
In some embodiments, the composition 950 can be dislodged from the wells 910 by flexing the mold apparatus 910, thereby deforming the well 910 shape and dislodging the composition 950.
In some embodiments, as shown in
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
The following materials and methods were used in the examples below, unless otherwise noted.
iPSC Differentiation
BU3 iPSC lines carrying the Nkx2.1-GFP and SPC-TdTomato reporters were obtained from Darrell N. Kotton, M.D. (4, 5). These cells were derived from a donor without known genetic abnormalities (11). This cell line had a normal karyotype by G-banding both before and after gene editing (5).
The differentiation of iPSCs was performed following previously published methods with modifications (4, 12). Briefly, BU3-NGST iPSCs that carry two fluorescent reporters for a lung epithelial progenitor marker and an alveolar type 2 cell marker, Nkx2.1-GFP and Surfactant Protein C (SPC)-TdTomato, respectfully, were maintained in mTESR medium (Stemcell Technologies, Vancouver, Canada). A stepwise differentiation procedure that mimics the lung developmental stages was initiated when cells reached 60-70% confluence. The basal medium for all differentiation steps was Dulbeco's Modified Eagle's Medium (DMEM)/F21 (Gibco, Waltham, Mass.) supplemented with B-27 (Gibco, Waltham, Mass.). First, the cells underwent definitive endodermal differentiation using the StemDiff kit (Stemcell Technologies, Vancouver, Canada) for 4 days followed by 4 days of 1 μM A8301 (Sigma, St. Louis, Mo.) and 1 μM IWR-1 (Sigma, St. Louis, Mo.) for anteriorized endodermal differentiation. The cells then underwent ventralized endodermal differentiation by exposing them to 10 ng/mL FGF-7 (Peprotech, Rocky Hill, N.J.), 10 ng/mL FGF-10 (Peprotech, Rocky Hill, N.J.), and 3 μM CHIR99021 (Tocris, Bristol, UK) for 7 days. After ventralization, the cells were stained with DAPI (Sigma, St. Louis, Mo.) and fluorescence-activated cell sorted (FACS) for purification of Nkx2.1-GFP positive cells.
Sorted Nkx2.1+ cells were embedded in 100% Matrigel (Corning, Corning, N.Y.) drops for the formation of alveolar spheres. Homogenous liquid precursor was aliquoted in 100 μL drops onto 12-well plastic culture plates. The culture medium for the formation, maintenance, and expansion (expansion media) of the alveolar spheres had the following composition: 50% Medium 199 (Life Technologies, Carlsbad, Calif.), 49% DMEM/F12 (Life Technologies, Carlsbad, Calif.), 2% fetal bovine serum (FBS) (Hyclone, Logan, Utah), B-27 (Life Technologies, Carlsbad, Calif.), 10 ng/mL FGF-7, 10 ng/mL FGF-10, 3 μM CHIR99021, 0.1 mM IBMX (Sigma, St. Louis, Mo.), 0.1 mM 8-Bromo-cAMP (Sigma, St. Louis, Mo.), 50 nM dexamethasone (Sigma, St. Louis, Mo.), 10 μM Y-27632 (Cayman Chemical, Ann Arbor, Mich.), and 50 μg/mL ascorbic acid (Stemcell Technology, Vancouver, Canada). The droplets were cultured for 7-14 days followed by Matrigel droplet digestion with Dispase (Corning, Corning, N.Y.). The remaining cellular spheres were trypsinized and FACS for GFP+TdTomato+ cells. These iPSC-AECs were used for further expansion.
Cadaveric Rat Lungs
All animal studies were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee Protocol #2014N000261 and conducted in accordance with The Guide for the Care and Use of Laboratory Animals. Rat lungs were explanted from outbred adult male Sprague-Dawley rats (300-400 g, Charles River Laboratories, Wilmington, Mass.). All rats were pair housed and given unrestricted access to chow and water prior to use. Animals were anesthetized with 5% isofluorane, a laparotomy was performed, heparin was administered intravascularly via the inferior vena cava, and the animal was sacrificed via exsanguination according to approved protocols. A sternotomy was then performed, and the lungs were explanted as previously described (13).
Floating Droplet Cell Culture
Matrigel with suspended cells was aliquoted into 100 μL drops containing approximately 20,000 cells each. Drops were placed in custom polydimethylsiloxane (PDMS) (Sigma, St. Louis, Mo.) molds (
Adherent Droplet Cell Culture
Matrigel based homogenous liquid precursor with suspended cells was aliquoted into 100 μL drops containing approximately 20,000 cells each. Drops were placed on tissue culture plastic in individual wells of a 12-well plate and allowed to gel at 37° C. for 20 minutes (
Rat Lung Decellularization
Cadaveric rat lungs were decellularized as previously described (13). Briefly, rat lungs were explanted from outbred adult male Sprague-Dawley rats. The pulmonary artery (PA) was cannulated via the right ventricular outflow tract, followed by tracheal cannulation. The lungs were perfused with a 0.1% sodium dodecyl sulfate (SDS) (Fisher Scientific, Waltham, Mass.) solution via the PA cannula for 2 hours. The lung scaffold was then perfused with sterile deionized water for 15 minutes followed by perfusion with 1% Triton X-100 (Fisher Scientific, Waltham, Mass.), all via the PA cannula. Finally, the decellularized lung scaffolds were washed with a minimum of 3 L phosphate buffered saline (PBS) over the subsequent 48 hours prior to use.
Seeding Lungs for Culture
After left pneumonectomy, rat lung scaffolds were mounted in custom bioreactors prefilled 100 mL alveolar sphere expansion medium perfused for a minimum of 1 hour at a flow rate of 1 mL/min in a 37° C., 5% CO2 incubator. Forty million iPSC-AECs were gravity seeded into the airway of each right lung with 50 mL expansion medium via the tracheal cannula. After seeding, the PA perfusion was paused for 90 minutes to allow for a static culture period promoting cell attachment to the scaffold. Perfusion was reinitiated at 1 mL/min for the next 16 hours, then increased to 3 mL/min for the remainder of the biomimetic culture period. Culture medium was changed every 48 hours for the 12-day culture period. Right upper and middle lobectomies were performed on post-seeding day 6. Tissue for RNA analysis was stored in Trizol (Fisher Scientific, Waltham, Mass.), and tissue for histologic analysis was fixed with 4% paraformaldehyde (PFA) (Westnet, Canton, Mass.) for 24 hours. On post-seeding day 12, the lower and accessory lobes were perfusion fixed via the tracheal cannula with 4% PFA for 24 hours.
Resazurin Assay
A resazurin cell metabolic assay was performed as previously described (7). Briefly, 80 mL spent media was mixed with PrestoBlue (Invitrogen, Waltham, Mass.) at a 1:20 dilution. Quadruplicate samples of the PrestoBlue mixture were saved in a 96-well flat bottom plate as controls. The mixture was then allowed to perfuse the biomimetic lung culture for 1 hour on experimental day 12. Upon completion, the spent media was sampled in quadruplicate and measured in a SpectraMax M3 multi-mode microplate reader (Molecular Devices, Sunnyvale, Calif.). The difference in fluorescence between the samples and controls was correlated to metabolic activity.
Histological Staining and Analysis
Alveolar spheres were embedded in Histogel (ThermoFisher, Waltham, Mass.) and paraffin-embedded prior sectioning. Fixed tissue sections were paraffin-embedded and sectioned. Tissue or cell sections mounted on glass slides were stained with hematoxylin and eosin for brightfield imaging. Tissue sections mounted on glass slides for immunofluorescent staining underwent antigen retrieval with a sodium citrate solution at high temperature and pressure and were permeabilized with 0.2% Triton X-100. Sections were then blocked with 10% fetal bovine serum (FBS) and 5% donkey serum (DS) (Sigma, St. Louis, Mo.). Primary antibodies were incubated overnight at 4° C. in a tris-buffered saline (TBS) with 0.5% DS solution then washed with TBS (1:50, Nkx2.1:ab72876, Abcam, Cambridge, UK; 1:200 SPC:ab3786, Abcam; 1:100 AQP5:ab92320, Abcam). Secondary antibodies were incubated for 2 hours at room temperature then washed with TBS (Alexa Fluor donkey anti-rabbit 594 or 647:ab150064 or ab150075, respectfully, Invitrogen). Slides were mounted with DAPI Fluoromount-G (Fisher Scientific, Waltham, Mass.). Images were captured using a Nikon Ti-PFS inverted microscope (Nikon, Tokyo, Japan). All fluorescent images for a given protein were captured with consistent exposure time and instrument gain. ImageJ software (National Institutes of Health, Bethesda, Md.) was used for analysis. Cell counts were obtained by isolating the DAPI color channel, subtracting the background signal, converting the image to a binary image, defining cell borders, and counting discrete nuclei. Image fluorescence was obtained by isolating the appropriate fluorescent channel, analyzing each image from a particular protein staining with consistent brightness and contrast, then calculating the mean fluorescence. Quantitative data was generated as a mean fluorescence per cell.
Spent Media Analysis from Biomimetic Lung Culture
The biomimetic lung culture media was changed every 48 hours and analyzed for pH, bicarbonate, lactate, and glucose concentration using an iSTAT (Abbott, Chicago, Ill.) point of care analyzer with CG4+cartridges (Abbott, Chicago, Ill.) and G cartridges (Abbott, Chicago, Ill.).
Relative Gene Expression Analysis
RNA was isolated by Trizol then reverse transcribed to cDNA by SuperScript Vilo Master Mix (Life Technologies, Carlsbad, Calif.). Gene expression was quantified by Taqman Assay with probes (see Key Resources Table for probe details) (Life Technologies, Carlsbad, Calif.) using the One Step Plus (Applied Biosystems, Foster City, Calif.) system. Gene expression was analyzed using the delta-delta method by normalizing to the housekeeping gene β-actin.
Flow Cytometric Analysis
Fluorescence activated cell sorting of Nkx2.1-GFP+/tdTomato+ cells was conducted using a FACSAria II (BD Biosciences, Franklin Lakes, N.J.). For phenotypic analysis, cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, N.J.) kit. Cells were stained with primary antibodies (1:250, SPC: ab40879, Abcam; 1:200 AQP5: ab92320, Abcam) for 30 minutes at 4° C., washed, then stained with secondary antibodies (1:200, Alexa Fluor donkey anti-rabbit 350 or 594: A10039 or ab150064, respectfully, Invitrogen) for 30 minutes at 4° C. Flow cytometric analysis was conducted using FlowJo software (BD Biosciences, Franklin Lakes, N.J.).
Statistical Methods
Data is presented as the mean±SEM. For tests of significance, a one-tailed Student's t-test with unequal variance was used to compare two populations. Significant was determined if p≤0.05. Data represented in FIGs. is assumed to be non-significant unless noted with an asterisk (*). For qPCR data, individual datapoints were excluded if they were determined to be greater than 3 standard deviations from the mean when including all datapoints in the descriptive statistic calculations. All statistical calculations were performed using Visual Basic for Applications.
To enable a floating droplet culture, we designed an autoclavable silicon mold (
The floating droplet culture method produced significantly more cells during the 8-day period than the adherent droplet culture method (2.86 million (M) cells/droplet vs 1.66 M cells/droplet, respectively, p<0.01,
Quantitative polymerase chain reaction (PCR) analysis demonstrated comparable expression of SPC between the adherent and floating culture methods but showed lower gene expression for Nkx2.1 in the floating droplet culture (p=0.041) (
Immunohistochemical staining of the lungs following biomimetic culture revealed a trend toward higher expression of Nkx2.1 in lungs with the adherent droplet culture cells (p=0.059) which corresponds with the PCR data from the end of the cell expansion period (
Spent media from the biomimetic lung culture demonstrated a comparable trend in bicarbonate change, lactate generation, and glucose consumption from both groups throughout the culture period (
To test if our scalable culture protocol is applicable for other human induced pluripotent stem cells derived alveolar epithelial cells (iPSC-AECs), we generate AECs from two other human iPSC lines. iPSC-17 cell line carries Nkx2.1-GFP and Surfactant Protein (SPC)-TdTomato reporters, while SPC2 cell line carried only SPC-TdTomato reporter. After 4 weeks of step-wised differentiation and 2 times of flow cytometry sorting, we purify SPC-TdTomato expressing AECs from iPSC-17 and SPC2 cell lines separately. 100% Matrigel is mixed with AECs then aliquoted into 100 μL drops containing approximately 20,000 cells each for the subsequent droplet or plate culture with 1 ml medium per drop, while medium is changed every other day.
Cells cultured for 8 days are harvested and counted for cell yield comparison between different methods, then fixed for flow cytometry analysis of AT2 cell marker SPC, AT1 cell marker AQP5, and lung epithelial progenitor marker Nkx2.1. Harvested cells are used for realtime PCR analysis and H&E staining of AEC marker genes. Data is generated from another two iPSC-17-AEC and SPC2-AECs to prove that our scalable method could promote proliferation while keeping AT2 phenotype in different iPSC-AEC cell lines.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/927,797, filed on Oct. 30, 2019, and 62/945,834, filed on Dec. 9, 2019. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/058350 | 10/30/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62945834 | Dec 2019 | US | |
| 62927797 | Oct 2019 | US |
