Universal tissue fabrication techniques for self-assembled organ production and regenerative medicine applications

Abstract

Methods and systems are described for a high-level approach to organ generation by self-assembly using bioprinting, overcoming longstanding challenges in replicating the intricate architecture and function of complex organs. In some embodiments, these methods are used to produce lung tissues and microtissues, demonstrating how mini-lung constructs can be produced on a small scale through specialized processes and media. In such embodiments, methods and systems for generating bioprinted lung tissues and microtissues are provided, as are tissues made by such methods. The tissues find use in implantation, drug discovery, personalized medicine, and other applications. Methods for the large-scale manufacturing of lung epithelial stem cells are also provided.

Description

[0001] UNIVERSAL TISSUE FABRICATION TECHNIQUES FOR SELF- ASSEMBLED ORGAN PRODUCTION AND REGENERATIVE MEDICINE APPLICATIONS CROSS REFERNCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Applications 63 / 851,080, filed 25 July 2025, and 63 / 694,095, filed 12 September 2024. The entireties of the above-referenced applications are incorporated herein by reference. FIELD

[0002] This disclosure relates to the fabrication of complex tissues and organs via the synergy of bioprinting and tissue self-assembly. BACKGROUND

[0003] The rapid and scalable creation of functional human tissues and organs remains a critical challenge in medicine, with the potential to dramatically transform healthcare through both implantable organs on demand and advanced in vitro models for disease research and drug testing. Current approaches, such as donor-based organ transplants, face significant limitations due to organ shortages and the persistent risk of immune rejection, necessitating ongoing immune management. Conventional in vitro cell culture methods also encounter challenges, including prolonged culture times, high costs, and difficulty in accurately reproducing complex tissue architectures necessary for authentic organ function. Xenotransplantation has also been proposed as a potential solution to provide tissues and organs for use in treating injury and disease. Here, gene editing can be used to reduce the immunogenicity of the animal-derived donor organs. However, maintaining the highly sterile environments required to rear these animals is expensive and challenging. SUMMARY

[0004] The present disclosure addresses the challenges faced in the prior art by providing novel bioprinting methodologies and cell culture modalities optimized for fabricating advanced tissue models. In some embodiments, these innovations are demonstrated through the creation of a complex 3D lung tissue model; in other embodiments, such innovations are used to create tissues and / or organs composed of repeating functional units or with layered, hierarchical, or branched structures.

[0005] Through the creation and development of state-of-the-art bioprinting technologies, specialized bioinks, and cell culture techniques, methods provided by the present disclosure enable the creation of highly functional 3D lung models. Compositions (e.g., lung models) provided by the present disclosure offer superior physiological relevancy and range of applicability as compared to similar compositions known in the art. While the prior art relied largely upon a monoculture of lung epithelial cells or lung organoids, the present disclosure significantly advances known technologies by providing a miniature lung having applicability in a wide variety of uses including, for example, drug discovery and regenerative medicine. In addition, the present disclosure provides, for the first time, long- term 2D culture of lung stem cells.

[0006] The innovative methods and compositions described herein represent a significant advancement in the field of tissue engineering. Embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of lung epithelial stem cells, studying lung biology and disease mechanisms, investigating viral infections, and enabling engineered lung implantation, among other advantages. In addition, the methods described in the present disclosure are also applicable to the fabrication of other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures.

[0007] The type of tissues and organs composed of repeating functional units that may be produced by the methods of the present disclosure can vary. Some embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of liver lobules, studying liver biology and disease mechanisms, investigating viral infections, and enabling engineered liver tissue implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of kidney nephrons, studying kidney biology and disease mechanisms, investigating viral infections, and enabling engineered kidney tissue implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestinal villi, studying intestinal biology and disease mechanisms, investigating viral infections, and enabling engineered intestine tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of vascularized cardiac tissue, studying cardiac tissue biology and disease mechanisms, investigating viral infections, and enabling engineered cardiac tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of pancreatic acini, studying pancreatic acinus biology and disease mechanisms, investigating viral infections, and enabling engineered pancreatic acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of islets of Langerhans, studying islet of Langerhans biology and disease mechanisms, investigating viral infections, and enabling engineered islet of Langerhans implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of seminiferous tubules, studying seminiferous tubule biology and disease mechanisms, investigating viral infections, and enabling engineered seminiferous tubule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovarian follicles, studying ovarian follicle biology and disease mechanisms, investigating viral infections, and enabling engineered ovarian follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of thyroid follicles, studying thyroid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered thyroid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of white pulp nodules, studying white pulp nodule biology and disease mechanisms, investigating viral infections, and enabling engineered white pulp nodule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of lymphoid follicles, studying lymphoid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered lymphoid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of osteons, studying osteon biology and disease mechanisms, investigating viral infections, and enabling engineered osteon implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of sarcomeres, studying sarcomere biology and disease mechanisms, investigating viral infections, and enabling engineered sarcomere implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of epidermal rete ridge units, studying epidermal rete ridge unit biology and disease mechanisms, investigating viral infections, and enabling engineered epidermal rete ridge unit implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of secretory acini, studying secretory acinus biology and disease mechanisms, investigating viral infections, and enabling engineered secretory acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0008] In one aspect, the present disclosure provides groundbreaking advancements in tissue engineering, specifically targeting the creation of advanced lung models using 3D bioprinting technology. In another aspect, the present disclosure provides a proprietary culture system that allows for the large-scale manufacturing of lung epithelial stem cells. In a third and fourth aspect, the present disclosure provides two distinct lung models: a layer- on-layer model and a 3D branching distal lung model. Methods of generating these two distinct models are also provided and, in some embodiments, comprise the creation of unique forms of fabricated lung microtissues, complex 3D branching distal lung-like tissue and layer-on-layer tissue, from human or animal-derived cells, including a heterogeneous population of bronchial stem cells, alveolar stem cells, endothelial cells, bronchial smooth muscle cells, lung fibroblast cells, and all the various differentiated lung subpopulation cells.

[0009] The layer-on-layer lung model provided by the present disclosure mimics the bronchoalveolar interface, a feat previously unachieved in tissue engineering. By encapsulating mesenchymal and vascular cells in a collagen-based bioink and employing sacrificial hydrogel-based walls, this model replicates the complex architecture of the distal lung. This is achieved, in part, by utilization of advanced bioprinting hardware, including a custom-built bioprinter, to ensure precise deposition and reliable assembly of the model, enabling enhanced reproducibility and cellular viability. In other embodiments, the methods describing the fabrication of the “layer-on-layer” model are also applicable to the fabrication of other tissues and organs with layered structures. Some embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0010] Similarly, the 3D branching distal lung model mirrors the native architecture of terminal bronchioles and air sacs, enabling the formation of bronchoalveolar junctions and supporting cell differentiation. This is a bioprinting process that utilizes specialized bioinks and an optimized bioprocess, facilitating the creation of intricate lung structures with unparalleled fidelity and functionality. Additionally, the methods describing the fabrication of the 3D branched model are also applicable to the fabrication of other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures. Some embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of liver lobules, studying liver biology and disease mechanisms, investigating viral infections, and enabling engineered liver tissue implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of kidney nephrons, studying kidney biology and disease mechanisms, investigating viral infections, and enabling engineered kidney tissue implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestinal villi, studying intestinal biology and disease mechanisms, investigating viral infections, and enabling engineered intestine tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of vascularized cardiac tissue, studying cardiac tissue biology and disease mechanisms, investigating viral infections, and enabling engineered cardiac tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of pancreatic acini, studying pancreatic acinus biology and disease mechanisms, investigating viral infections, and enabling engineered pancreatic acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of islets of Langerhans, studying islet of Langerhans biology and disease mechanisms, investigating viral infections, and enabling engineered islet of Langerhans implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of seminiferous tubules, studying seminiferous tubule biology and disease mechanisms, investigating viral infections, and enabling engineered seminiferous tubule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovarian follicles, studying ovarian follicle biology and disease mechanisms, investigating viral infections, and enabling engineered ovarian follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of thyroid follicles, studying thyroid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered thyroid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of white pulp nodules, studying white pulp nodule biology and disease mechanisms, investigating viral infections, and enabling engineered white pulp nodule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of lymphoid follicles, studying lymphoid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered lymphoid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of osteons, studying osteon biology and disease mechanisms, investigating viral infections, and enabling engineered osteon implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of sarcomeres, studying sarcomere biology and disease mechanisms, investigating viral infections, and enabling engineered sarcomere implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of epidermal rete ridge units, studying epidermal rete ridge unit biology and disease mechanisms, investigating viral infections, and enabling engineered epidermal rete ridge unit implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of secretory acini, studying secretory acinus biology and disease mechanisms, investigating viral infections, and enabling engineered secretory acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0011] Both the 3D branching model and layer-on-layer model are vascularized, with 3D branching vasculature being present around the alveoli and throughout the mesenchymal regions. The lung microvasculature plays a role in cell signaling, nutrient transport, and oxygenation. The vessels help immature stem cells proliferate and aid in their differentiation as well. Additionally the vessels prevent the formation of necrotic tissue in the center of the models, bolstering physiological relevancy, longevity, and ease of use. Additionally, the vessels enable researchers to observe the pathology of respiratory infections and effects of drugs by observing the air-liquid interface of the alveoli and its surrounding vasculature.

[0012] Both the 3D branching model and layer-on-layer model can be achieved by methods provided by the present disclosure that allow for the production of a plurality of lung tissues. In various embodiments, the methods comprise: culturing progenitor cells, primary cells, and / or cell lines in a hydrogel using “lung initiation medium” and “lung maturation medium” (both as described herein), seeding the progenitor cells, primary cells, and / or cell lines into a cell culture device comprising at least one compartment comprising a plurality of microwells and / or a permeable membrane insert; culturing the cells in the culture device under conditions suitable to allow the cells to form vascularized lung microtissues; and culturing the formed lung microtissues.

[0013] Formation and maturation of both of these engineered tissues are facilitated by a culture system (disclosed herein) that allows for the large-scale manufacturing of lung epithelial stem cells. In this regimen, various growth factors and nutrients are supplemented into a tissue culture medium and used in conjunction with an air-liquid interface culture system allowing the close recapitulation of lung structures.

[0014] In various embodiments, the culture system provides for methods of manufacturing lung epithelial stem cells, the methods comprising: coating tissue culture vessels with extracellular matrix proteins including but not limited to laminin, collagen, entactin / nidogen, and heparin sulfate proteoglycans; and using said coated tissue culture vessels to manufacture lung epithelial cells in 2 dimensions, and to derive and manufacture alveolar and bronchial stem cells long-term, among other things. The disclosed methods include the use of cell culture media containing agonists for Wnt / β-catenin, EGFR, FGFR, VEGFR signaling and antagonists for GSK3, BMP, p38 MAPK, ROCK, and TGFβ signaling pathways.

[0015] The proprietary culture system provided by the present disclosure prolongs the number of passages multipotent lung stem cells are capable of undergoing, without losing multipotency. This can be achieved, for example, by coating a tissue culture vessel or bioreactor with extracellular matrix proteins including but not limited to collagen, laminin, entactin / nidogen, and heparin sulfate proteoglycans. Passages up to at least passage 8 show no signs of differentiation. In some embodiments, the total number of passages is 8 – 20, in some embodiments 8 – 18, in some embodiments, 8-16, in some embodiments 8-14, in some embodiments 8-12, and in some embodiments 8-10 passages.

[0016] Methods, models and systems provided by the present disclosure represent a paradigm shift in tissue engineering, offering researchers unprecedented capabilities to model complex lung biology, study disease mechanisms, and explore therapeutic strategies in vitro. Additionally, the proprietary culture system will facilitate the rapid and large scale manufacturing of lung epithelial cells needed to supply the nascent lung replacement industry. Suitable uses for these methods, models, and / or systems include, but are not limited to, drug development, drug screening, synthetic biology, biodefense studies, disease modeling, developmental studies, organ engineering, and organ replacement. In addition, the methods described in the present disclosure are also applicable to the fabrication of other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures. Some embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of liver lobules, studying liver biology and disease mechanisms, investigating viral infections, and enabling engineered liver tissue implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of kidney nephrons, studying kidney biology and disease mechanisms, investigating viral infections, and enabling engineered kidney tissue implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestinal villi, studying intestinal biology and disease mechanisms, investigating viral infections, and enabling engineered intestine tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of vascularized cardiac tissue, studying cardiac tissue biology and disease mechanisms, investigating viral infections, and enabling engineered cardiac tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of pancreatic acini, studying pancreatic acinus biology and disease mechanisms, investigating viral infections, and enabling engineered pancreatic acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of islets of Langerhans, studying islet of Langerhans biology and disease mechanisms, investigating viral infections, and enabling engineered islet of Langerhans implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of seminiferous tubules, studying seminiferous tubule biology and disease mechanisms, investigating viral infections, and enabling engineered seminiferous tubule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovarian follicles, studying ovarian follicle biology and disease mechanisms, investigating viral infections, and enabling engineered ovarian follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of thyroid follicles, studying thyroid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered thyroid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of white pulp nodules, studying white pulp nodule biology and disease mechanisms, investigating viral infections, and enabling engineered white pulp nodule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of lymphoid follicles, studying lymphoid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered lymphoid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of osteons, studying osteon biology and disease mechanisms, investigating viral infections, and enabling engineered osteon implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of sarcomeres, studying sarcomere biology and disease mechanisms, investigating viral infections, and enabling engineered sarcomere implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of epidermal rete ridge units, studying epidermal rete ridge unit biology and disease mechanisms, investigating viral infections, and enabling engineered epidermal rete ridge unit implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of secretory acini, studying secretory acinus biology and disease mechanisms, investigating viral infections, and enabling engineered secretory acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0017] It is thus one object of the present disclosure to provide an approach to overcome the lack of 3D lung microtissues with multiple cell types.

[0018] It is another object of the present disclosure to provide an approach suitable for 3D tissue culture which helps to promote distal lung-like structure formation.

[0019] It is another object of the present disclosure to provide an approach suitable for 3D tissue culture which mimics the layered structure of lung tissue found in vivo.

[0020] It is another object of the present disclosure to provide an approach suitable for 3D tissue culture which helps to improve the functionality, viability and stability of 3D tissues.

[0021] It is another object of the present disclosure to provide an approach suitable for 2D tissue culture of lung epithelial stem cells which helps to improve the accessibility, quantity and cost of 3D tissues created with said cells.

[0022] In some embodiments, lung microtissues provided by the present disclosure exhibit ciliary beating.

[0023] In some embodiments, lung microtissues provided by the present disclosure exhibit production of mucus.

[0024] In some embodiments lung microtissues provided by the present disclosure produce pulmonary surfactant.

[0025] In some embodiments, lung microtissues provided by the present disclosure comprise bronchial stem cells, alveolar stem cells, endothelial cells, bronchial smooth muscle cells, lung fibroblast cells, and / or any one or more of the remaining differentiated lung subpopulations of cells.

[0026] In some embodiments, lung microtissues provided by the present disclosure are about 5 mm – about 50 cm in height and / or width and / or length, wherein the height, width and / or length are each measured independently of each other.

[0027] In some embodiments, complex 3D branched lung microtissues provided by the present disclosure can comprise bronchial stem cells, alveolar stem cells, endothelial cells, bronchial smooth muscle cells, lung fibroblast cells, and / or any one or more of the various differentiated lung subpopulations of cells.

[0028] In some embodiments, complex 3D branched microtissues provided by the present disclosure can comprise the cell types of any other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures.

[0029] In some embodiments, complex 3D branched lung microtissues provided by the present disclosure are about 5 mm – about 50 cm in height and / or width and / or length, wherein the height, width and / or length are each measured independently of each other.

[0030] In some embodiments, layered 3D lung microtissues provided by the present disclosure can comprise bronchial stem cells, alveolar stem cells, endothelial cells, bronchial smooth muscle cells, lung fibroblast cells, and / or any one or more of the various differentiated lung subpopulations of cells.

[0031] In some embodiments, layered 3D microtissues provided by the present disclosure can comprise the cells of any other tissues and organs with layered structures.

[0032] In some embodiments, layered 3D lung microtissues provided by the present disclosure are about 5 mm – about 50 mm in height and / or width and / or length, wherein the height, width and / or length are each measured independently of each other.

[0033] In another aspect, the disclosure relates to methods for viral pathology studies using lung microtissues generated by the methods of the disclosure.

[0034] In another aspect, the disclosure relates to methods for drug discovery using lung microtissues generated by the methods of the disclosure.

[0035] In another aspect, the disclosure relates to methods for drug screening studies using lung microtissues generated by the methods of the disclosure.

[0036] In another aspect, the disclosure relates to methods for organ implantation or replacement using lung microtissues generated by the methods of the disclosure.

[0037] In another aspect, the disclosure relates to methods for synthetic biology using lung microtissues generated by the methods of the disclosure.

[0038] In another aspect, the disclosure relates to methods for biodefense studies using lung microtissues generated by the methods of the disclosure.

[0039] In another aspect, the disclosure relates to methods for developmental studies using lung microtissues generated by the methods of the disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Bioprinting and tissue self-assembly process. (A) A mixture of stem cells and differentiated cells are positioned in 3D space using bioprinting. (B) The cells differentiate and self-assemble into tissue structures. (C) Complex tissue structures composed of repeating units and containing multiple cell types are formed.

[0041] Figure 2 illustrates a bioprinted lung tissue construct produced according to embodiments provided herein, showing the different cell types and structural components. The figure depicts bronchial cells (2) in the outer compartment, a sacrificial wall (4) made from a hydrophilic non-ionic copolymer surfactant (Pluronic F-127, CAS No.9003-11-6, in the depicted embodiment), alveolar cells (6) in the inner compartment, and a mixture of fibroblasts and endothelial cells in Type I collagen (8). The support structure (10) secures the basal layer, and the bronchial-alveolar interface (12) is depicted. The permeable membrane (14) and the wall of the permeable membrane insert (16) are also shown, with the bronchial monolayer (18) and alveolar monolayer (20) clearly indicated.

[0042] Figure 3 presents a detailed view of a lung tissue construct produced according to embodiments provided herein. The depicted embodiment is similar to FIG. 2, but with an emphasis on the arrangement and interaction of the different cell types and structural elements. Bronchial cells (2) are situated in the outer compartment, with the sacrificial wall (4) made from a hydrophilic non-ionic copolymer surfactant (Pluronic F127, CAS No.9003- 11-6, in the depicted embodiment) and alveolar cells (6) in the inner compartment. The fibroblasts and endothelial cells in Type I collagen (8), support structure (10), bronchial- alveolar interface (12), permeable membrane (14), wall of the permeable membrane insert (16), bronchial monolayer (18), and alveolar monolayer (20) are all highlighted.

[0043] Figures 4a and 4b are diagrams illustrating one embodiment of a workflow for a layer-on-layer bioprocess and culture according to embodiments provided herein. Figure 4a illustrates the steps to assemble a lung model up until the point that it is left to culture. Figure 4b is a timeline for culturing a lung model after it has been assembled, showing the media type and placement for the two phases of culturing. The composition of both initiation and differentiation / maturation media are provided herein.

[0044] Figures 5a, 5b, 5c, and 5d depict images of the epithelial layer of a layer-on-layer model produced according to embodiments provided herein. Figure 5a is an image of the alveolar cell monolayer at day 1. Figure 5b is an image of the confluent alveolar cell monolayer. Figure 5c is an image of a bronchial cell monolayer at day 1. Figure 5d is an image of the confluent bronchial cell monolayer.

[0045] Figures 6a and 6b provide immunostained images of the pluronic wall interface mechanism and maturation of the layer-on-layer epithelial system produced according to embodiments provided herein. Figure 6a is a stitched top-view image of a whole 12-well transwell insert showing the separation between Krt5+ / Hoechst+ basal cells from the alveolar cells (Krt5- / Hoechst+). Figure 6b provides images taken at the different denoted areas. In the lung model depicted, the Krt5+ basal cells are mostly situated in the bronchial region while HT1-56+ alveolar cells are predominantly observed in the middle alveolar region. Tight junction staining (ZO-1) is observed in all three regions.

[0046] Figures 7a, 7b, 7c, 7d, 7e, and 7f provide immunofluorescence images of the layer- on-layer model for various distal lung cell markers produced according to embodiments provided herein. Figures 7a – 7e are 10 µm frozen OCT sections (cross-sections) stained for a) E-cadherin (epithelial cells), UEA1 (endothelial cells), b) MUC5AC (Goblet cells), c) acetylated tubulin (ciliated cells), d) uteroglobin (CC10, club cells), and e) mesenchymal cells (Vimentin+). Figure 7f is of alveolar differentiation markers for type I (HT1-56) and type II (Pro-SPC) alveolar cells. Hoechst3342 was used as a nuclear stain in all the images presented.

[0047] Figures 8a, 8b, 8c, and 8d provide images showing the results of a live-dead viability assay of the layer-on-layer system using a green fluorescent cell marker dye (Calcein AM) and a dimeric cyanine nucleic acid stain (BOBOTM-3 iodide, Thermo Fisher Scientific Inc., USA). Figures 8a and 8b show day 21 of a layer-on-layer model demonstrating highly positive staining for the green fluorescent stain with sparse staining for the dimeric cyanine nucleic acid stain in both alveolar and bronchial regions. Figures 8c and 8d show a 100% Dead control obtained by exposing a day 21 layer-on-layer lung model to 70% ethanol for 30 min at 37°C.

[0048] Figure 9 illustrates the components of one embodiment of the 3D Branching Distal Lung Model provided by the present disclosure, including a wall of a permeable membrane insert (16), fibroblasts and endothelial cells in Type I collagen and a solution of extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA) (8), alveolar cells (6) represented by dots, bronchial cells (2) represented by lines, and the permeable membrane (14).

[0049] Figures 10a, 10b, and 10c depict one embodiment of a workflow of the bioprocess and culture of the 3D branching distal lung model provided herein. Figure 9a shows the bioprocess workflow for the 3D branching lung model featuring endothelial seeding and bioprinting in a mesenchyme infill. Figure 9b is a timeline showing 5 days of submerged culture during initiation and maturation in air-liquid interface (ALI). The composition of both initiation and differentiation / maturation media are provided herein. Figure 9c is a brightfield image of a branching lung model at day 9 of culture. The zoom box (right) highlights the bronchial (bro), alveolar (alv), and the mesenchymal (mes) regions of the tissue.

[0050] Figures 11a, 11b, 11c, 11d, and 11e provide images showing the results of a live- dead viability staining of the 3D bioprinted branching lung structure using a green fluorescent cell marker dye (Calcein AM) and a dimeric cyanine nucleic acid stain (BOBOTM-3 iodide, Thermo Fisher Scientific Inc., USA) at day 21 post-print. Figures 11a and 11b show alveolar air-sacs, Figure 11c shows bronchial cells, Figure 11d shows mesenchymal region, and Figure 11e is a dead control.

[0051] Figures 12a, 12b, 12c, 12d, 12e, and 12f provide images validating cell differentiation and tissue maturation in the 3D bioprinted branching lung tissues. Distal lung cell markers were stained in day 21 cryosections (10 µm, in OCT). Figure 12a shows ciliated cells populating the bronchial lumen, Figure 12b shows mucin expression in cells near the bronchial lumen, Figure 12c shows that club cell marker Uteroglobin (CC10) is expressed and secreted in the bronchial lumen, Figure 12d is a bronchoalveolar junction formation showing fusion of the alveolus and the bronchiole, Figure 12e shows epithelial and endothelial interaction and maturation of both systems as confirmed by ZO-1 (tight junction) staining, And Figure 12f depicts alveolar structure and cell differentiation, showing alveolar type I cells (AT1, HT1-56+ lining, cyan arrows) and alveolar type 2 (AT2, Pro-SPC+ cells, red arrows) in differentiated airsac-like structures. (int = interstitium; lumen = airsac lumen)

[0052] Figures 13a and 13b provide images confirming infectivity of the PR8-H1N1- mCherry influenza virus in layer-on-layer model’s bronchial and alveolar sections. Figure 13a shows images of an uninfected layer-on-layer model after day 1 and day 2. There was no viral activity detected. In the model provided in Figure 13b, influenza viruses were diluted to 1:100 in a total of 1 mL of DMEM media and applied to day 21 layer-on-layer models. Infection was monitored using a fluorescent microscope at 24 and 48 h post- infection and presence of mCherry+ cells was observed denoting effective viral infection in both the alveolar and the bronchial compartments.

[0053] Figures 14a and 14b provide images of the branching model at different times after printing. Figure 14a shows an image of a freshly printed branching model at day 0 taken with a 4K color inverted microscope. Figure 14b is a whole-stitched brightfield image of the branching model at day 18 taken with a fluorescence microscope (e.g., Olympus, Evident Corporation, Tucson, AZ, USA). DETAILED DESCRIPTION

[0054] The present disclosure provides significant advances in 3D bioprinting and tissue engineering. To overcome the barriers existing in the prior art, the present invention introduces an innovative tissue engineering methodology that integrates the spatial precision of 3D bioprinting with cellular self-assembly capabilities. Unlike traditional methods which rely on slow maturation from a limited initial cell population, this novel approach employs a three-stage process by (1) facilitating high-volume cell growth outside the tissue construct, (2) precisely printing these cells in three dimensions, and (3) leveraging self-assembly within the printed environment to form sophisticated microarchitectures.

[0055] This invention is first demonstrated through the production of lung tissue, specifically the intricate distal lung architecture comprising bronchioles and alveolar structures. Unlike traditional 2D cultures or animal-derived lung models, which often inadequately represent human lung complexity, this method reliably generates physiologically accurate lung tissue, enabling robust in vitro research applications, significantly enhancing studies of lung diseases, drug effects, and therapeutic strategies.

[0056] Though initially validated with lung tissue, the invention explicitly encompasses broader applications across various organs characterized by repeating structural units, such as liver lobules, kidney nephrons, intestinal villi, or cardiac tissues with vascular networks. This generalized applicability provides the basis for a transformative shift in tissue engineering, offering both scalable, implantable organ solutions and sophisticated models for biomedical research.

[0057] In summary, this patent addresses critical limitations in current tissue engineering strategies by merging high-volume cell expansion, precise bioprinting technology, and innate cellular self-assembly, establishing a versatile and rapid pathway to generating functional human tissues and organs for clinical and research applications.

[0058] The disclosed methods herein significantly advance 3D bioprinting and tissue engineering, and deliver highly sophisticated functional tissues that recapitulate natural human tissues and organs. These methods combine large-scale cell expansion in bioreactors, 3D printing of cells, and natural tissue self-assembly processes, while reducing production times and costs. The disclosed techniques can be broadly applied to any organ with repeating functional units or layered, hierarchical, or branched architectures (e.g., liver, kidney, heart, or intestine), although they have been applied to the production of lung tissue in the present disclosure as an example.

[0059] By harnessing the power of large-scale cell expansion in bioreactors, the three- dimensional (3D) arrangement of specific cell types, and natural cell and tissue self- assembly processes, the methods disclosed herein expedite and reduce the cost of producing functional tissue. Although the techniques are broadly applicable to any organ composed of repeating functional units or layered, hierarchical, or branched architectures (e.g., liver, kidney, heart, or intestine), an illustrative example is provided for creating “mini-lungs” as a proof-of-concept. The resulting tissues find applications in drug discovery, disease modeling, personalized medicine, regenerative therapies, and on-demand organ replacement.

[0060] In a first aspect, the present disclosure provides a “layer-on-layer” model of engineering tissue that mimics the layered structure found in the distal lung. Grown with stem cells, endothelial cells, and mesenchymal cells, this “layer-on-layer” model produces tissues having an accurate representation of the distal lung architecture. Layer on layer model tissues are suitable for a wide range of applications including, for example, use in viral pathophysiology studies, drug discovery, personalized therapy, and others. The layered lung tissue produced by this “layer-on-layer” model may find use as lung grafts to restore lung function. Additionally, the methods describing the fabrication of the “layer-on-layer” model are also applicable to the fabrication of other tissues and organs with layered structures. Some embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0061] In a second aspect, the present disclosure provides a three-dimensional (3D) branched model of engineering tissue that mimics the in vivo structures found in the distal lung, for example in the human distal lung. Grown with stem cells, endothelial cells, and mesenchymal cells, this 3D branched model produces tissues having an accurate representation of the distal lung’s 3D architecture. Tissues engineered according to this model are suitable for a wide range of applications including, for example, use in viral pathophysiology studies, drug discovery, personalized therapy, and others. The 3D branching lung tissue produced according to this 3D branched model may find use as lung grafts to restore lung function. Engineered 3D branching lung models may also be used in lung transplant situations. Additionally, the methods describing the fabrication of the 3D branched model are also applicable to the fabrication of other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures. Some embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of liver lobules, studying liver biology and disease mechanisms, investigating viral infections, and enabling engineered liver tissue implantation, among other advantages. Other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of kidney nephrons, studying kidney biology and disease mechanisms, investigating viral infections, and enabling engineered kidney tissue implantation, among other advantages. Still other embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestinal villi, studying intestinal biology and disease mechanisms, investigating viral infections, and enabling engineered intestine tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of vascularized cardiac tissue, studying cardiac tissue biology and disease mechanisms, investigating viral infections, and enabling engineered cardiac tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of pancreatic acini, studying pancreatic acinus biology and disease mechanisms, investigating viral infections, and enabling engineered pancreatic acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of islets of Langerhans, studying islet of Langerhans biology and disease mechanisms, investigating viral infections, and enabling engineered islet of Langerhans implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of seminiferous tubules, studying seminiferous tubule biology and disease mechanisms, investigating viral infections, and enabling engineered seminiferous tubule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovarian follicles, studying ovarian follicle biology and disease mechanisms, investigating viral infections, and enabling engineered ovarian follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of thyroid follicles, studying thyroid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered thyroid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of white pulp nodules, studying white pulp nodule biology and disease mechanisms, investigating viral infections, and enabling engineered white pulp nodule implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of lymphoid follicles, studying lymphoid follicle biology and disease mechanisms, investigating viral infections, and enabling engineered lymphoid follicle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of osteons, studying osteon biology and disease mechanisms, investigating viral infections, and enabling engineered osteon implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of sarcomeres, studying sarcomere biology and disease mechanisms, investigating viral infections, and enabling engineered sarcomere implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of epidermal rete ridge units, studying epidermal rete ridge unit biology and disease mechanisms, investigating viral infections, and enabling engineered epidermal rete ridge unit implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of secretory acini, studying secretory acinus biology and disease mechanisms, investigating viral infections, and enabling engineered secretory acinus implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skin, studying skin biology and disease mechanisms, investigating viral infections, and enabling engineered skin implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of blood vessel walls, studying blood vessel wall biology and disease mechanisms, investigating viral infections, and enabling engineered blood vessel wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of intestine wall, studying intestinal wall biology and disease mechanisms, investigating viral infections, and enabling engineered intestinal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of stomach tissue, studying stomach tissue biology and disease mechanisms, investigating viral infections, and enabling engineered stomach tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of esophagus wall, studying esophageal wall biology and disease mechanisms, investigating viral infections, and enabling engineered esophagus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of tracheal wall, studying tracheal wall biology and disease mechanisms, investigating viral infections, and enabling engineered tracheal wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of urinary bladder wall, studying urinary bladder wall biology and disease mechanisms, investigating viral infections, and enabling engineered urinary bladder wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of uterus wall, studying uterine wall biology and disease mechanisms, investigating viral infections, and enabling engineered uterus wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cornea, studying corneal biology and disease mechanisms, investigating viral infections, and enabling engineered cornea implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of retina, studying retinal biology and disease mechanisms, investigating viral infections, and enabling engineered retina implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large- scale manufacturing of heart wall, studying heart wall biology and disease mechanisms, investigating viral infections, and enabling engineered heart wall implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of meninges, studying meningeal biology and disease mechanisms, investigating viral infections, and enabling engineered meninges implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of cerebral cortex, studying cortical biology and disease mechanisms, investigating viral infections, and enabling engineered cerebral cortex implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of skeletal muscle, studying skeletal muscle biology and disease mechanisms, investigating viral infections, and enabling engineered skeletal muscle implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of testis tissue, studying testicular tissue biology and disease mechanisms, investigating viral infections, and enabling engineered testis tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of ovary tissue, studying ovarian tissue biology and disease mechanisms, investigating viral infections, and enabling engineered ovary tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of placenta tissue, studying placental tissue biology and disease mechanisms, investigating viral infections, and enabling engineered placenta tissue implantation, among other advantages. Still further embodiments provided by the present disclosure offer new opportunities for large-scale manufacturing of the renal filtration barrier of the kidney, studying renal filtration barrier biology and disease mechanisms, investigating viral infections, and enabling engineered filtration barrier implantation, among other advantages.

[0062] In a third aspect, the present disclosure provides a method for upscaling lung epithelial stem cells. This method combines the use of extracellular matrix proteins in unique culture conditions, allowing for inexpensive and easy manufacture of large numbers of cells. The disclosed method preserves the immature morphology into passages in excess of passage 8, thereby increasing the total yield of cells, and reducing associated costs significantly. Definitions

[0063] As used herein, the following terms shall have the following meanings, unless the context expressly states, or clearly requires, otherwise.

[0064] The terms “about,” “approximately,” etc., when used in relation to numerical limitations or ranges, mean that the recited limitation or range may vary by up to 10%. For example, “about 750” can mean as little as 675 or as much as 825, or any value therebetween. When used in relation to ratios or relationships between two or more numerical limitations or ranges, the terms “about,” “approximately,” etc. mean that each of the limitations or ranges may vary by up to 10%; for example, a statement that two quantities are “approximately equal” can mean that a ratio between the two quantities is as little as 0.9:1.1 or as much as 1.1:0.9 (or any value therebetween), and a statement that a four-way ratio is “about 5:3:1:1” can mean that the first number in the ratio can be any value of at least 4.5 and no more than 5.5, the second number in the ratio can be any value of at least 2.7 and no more than 3.3, and so on.

[0065] The phrase ”air-liquid interface” (“ALI”) refers to a culture system used to grow lung microtissues where the tissue is exposed to air on one side and liquid culture medium on the other. Generally speaking, this method mimics the natural environment of lung tissue and thereby, in some embodiments, promotes the differentiation and maturation of lung epithelial cells. ALI can be used to facilitate the development of functional lung models disclosed herein, for example either or both of the layer-on-layer and 3D branching distal lung models described herein. By providing an ALI, a culture system can be utilized to support the formation of physiologically relevant structures, such as bronchoalveolar junctions, and to enhance the functional properties of lung microtissues by, for example, making them suitable for applications in disease modeling, drug testing, and therapeutic research.

[0066] The term ”bioink” refers to any biocompatible material capable of use in 3D bioprinting to fabricate tissue structures and organoids, including lung microtissues. In the context of lung models, bioink typically includes a mixture of extracellular matrix proteins, such as collagen, laminin, entactin / nidogen, and heparan sulfate proteoglycans, combined with lung epithelial stem cells, mesenchymal cells, and vascular cells, among other things. This composition allows the bioink to support cell viability, proliferation, and differentiation, replicating the complex architecture and functionality of lung tissue.

[0067] “Distal lung” means the region of the lung that includes the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli. This area is primarily responsible for gas exchange, where oxygen enters the blood and carbon dioxide is removed. The distal lung is an essential focus due to its critical role in respiratory function and its susceptibility to various diseases and infections.

[0068] The terms “lung microtissue,” “lung microstructure,” and “lung model,” which are used interchangeably in this disclosure, refer to engineered constructs that replicate specific aspects of lung tissue organization and function. Lung microstructures typically comprise multiple cell types found in the lung, for example bronchial epithelial cells, alveolar epithelial cells, endothelial cells, mesenchymal cells, and others. Lung microtissue constructs are generated using 3D cell culture techniques, often in hydrogel matrices, and provide a more physiologically relevant platform for studying cell-cell interactions, tissue development, and disease processes compared to traditional 2D cell culture models.

[0069] Other features and advantages of the invention will be apparent from the following detailed description, the figures, and from the claims. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the disclosure. Layer-On-Layer Method and Model

[0070] The layer-on-layer model is an engineered tissue designed to recapitulate the layered structure found in the distal lung. In various embodiments this lung model comprises an epithelial layer and an interstitial layer, modeling the fibrous-cartilaginous and epithelial layering found in the human lung. Grown with stem cells, endothelial cells, and mesenchymal cells, this model provides an accurate representation of the distal lung’s architecture.

[0071] Generally speaking, this layer-on-layer lung model is produced from two populations of lung epithelial stem cells that are grown in separate regions on a permeable membrane, with an interstitial layer just below containing fibroblasts and endothelial cells encapsulated in a 3D matrix. Bronchial stem cells are grown on an outer region, and alveolar stem cells are grown on an inner region, with both lung populations being on the same plane (see, e.g., Figures 2, 3). In some embodiments, the two lung populations are seeded onto a permeable membrane that has been previously coated with extracellular matrix proteins, and are initially divided by a sacrificial wall. Following the sacrifice and / or removal of the wall, the two lung populations form a bronchoalveolar junction (see, e.g., Figure 6a). Optionally, an air-liquid interface is created by removing growth medium from the top surface of a permeable membrane insert during culture, helping to mature the tissue and provide a route of entry to the apical membrane.

[0072] Methods for the production of a layer-on-layer lung model includes steps such as permeable membrane coating, wall printing, and cell seeding, all optimized to maximize model fidelity and cellular viability. The culture regimen transitions from initiation to maturation phases, involving specific media formulations and air-liquid interface (ALI) culturing techniques to promote epithelial differentiation and model maturation.

[0073] The layer-on-layer model serves as a sophisticated mimicry of the bronchoalveolar interface. Components of this model include mesenchymal and vascular cells encapsulated within a bioink (referred to as the “basal layer”), facilitated by a sacrificial wall. The resulting architecture replicates the intricate morphology of the distal lung, enabling comprehensive study of pulmonary physiology and pathology.

[0074] Additionally, the methods describing the fabrication of the “layer-on-layer” model are also applicable to the fabrication of other tissues and organs with layered structures. One non-limiting embodiment is the structure of the intestine where the basal layer includes mesenchymal and vascular cells encapsulated within a bioink and the permeable membrane insert is covered with intestinal mucosal cells, progenitors or stem cells. Another non- limiting embodiment is the structure of the liver where the basal layer includes mesenchymal and vascular cells encapsulated within a bioink and the permeable membrane insert is covered with hepatocytes and cholangiocytes, or their associated progenitors or stem cells.

[0075] One non-limiting embodiment of a method of producing a layer-on-layer lung model is as follows. Permeable Membrane Coating

[0076] In various embodiments, the construction of the layer-on-layer lung model begins with the coating of a permeable membrane with extracellular matrix proteins diluted in cell medium. The permeable membrane can be of any configuration that allows for accommodation of a sacrificial wall and for the seeding of two different populations of lung stem cells. For example, the membrane may be a “stand-alone” component or it may be integrated into a support structure that is suitable for insertion into a tissue culture well. The permeability of the membrane can vary, to suit the needs of the lung model, and may range from a pore size of 0.4 μm – 8.0 μm (e.g., Falcon and / or Transwell Permeable Supports, Corning Incorporated, Glendale, AZ, USA). In some embodiments, such as that depicted in Figure 4a, the layer-on-layer model uses a well plate and compatible permeable membrane insert. In such embodiments, the permeable membrane insert sits on top of a standardized 12-well plate, with each insert extending into an individual well (Figure 4a), though any suitable culture vessel may be used, for example 6-well permeable membrane inserts and plates, 12-well permeable membrane inserts and plates, 24-well permeable membrane inserts and plates, 48-well permeable membrane inserts and plates, bioreactors, industrial vats, and so on. The configuration of the permeable insert – well combination is not particularly limited and may be adjusted to suit the desired size of the lung model.

[0077] Once a suitable configuration of an insert – well combination is selected, the permeable membrane is coated with extracellular matrix (ECM) proteins. The exact composition of the extracellular matrix proteins can vary, depending on the desired nature of the lung model being created, though in some embodiments the composition of the extracellular matrix proteins is similar to solubilized extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA). In various embodiments, the extracellular matrix proteins are selected from laminin, collagen, entactin / nidogen, heparan sulfate proteoglycans, and combinations of any of the foregoing. The proteins are dissolved in a suitable solvent, such as tissue culture media (e.g., DMEM medium, Basal medium, or the like), initiation medium (described herein), or other suitable liquid. The concentration of each extracellular matrix protein in the suitable solvent can vary. In some embodiments, the concentration of each ECM protein in solution is independently about 0.1 % – about 25 %. In some embodiments, the total concentration of all ECM proteins in the suitable solvent is 1 %, 2 %, 5 %, 10 %, 15 %, 20 %, or 25 %.

[0078] This extracellular matrix protein solution is placed into contact with those surfaces of the membrane that are to be coated for a period of time sufficient for the extracellular matrix proteins to adhere to the surfaces, for example from 1 minute to 2 days’ time. Thereafter, excess coating solution is removed, for example by aspiration or other suitable methods, leaving the coated permeable membrane.

[0079] In other embodiments, the permeable membrane is not coated with extracellular matrix proteins. In such embodiments, the foregoing step is omitted and the method of generating a layer-on-layer lung model proceeds with placement of a sacrificial wall. Sacrificial Wall

[0080] The layer-on-layer lung model includes deposition of a sacrificial wall following the coating of the permeable membrane insert (if coating is applicable or desired). The sacrificial wall serves to keep the two types of lung stem cells separated from each other during seeding (see, e.g., Figures 2a, 2b). In various embodiments, the sacrificial wall is deposited directly onto the permeable membrane insert using a computer-controlled deposition device (e.g., a bioprinter). The sacrificial wall divides the surface of the permeable membrane into inner and outer regions. In some embodiments, the permeable membrane insert can be placed into the deposition device / bioprinter, where the sacrificial wall can be deposited onto it directly.

[0081] The composition of the sacrificial wall can vary, though it will typically be formulated out of materials that will not interfere with the seeding or initial culturing processes. In some embodiments, the sacrificial wall is deposited using a bioink comprising a block copolymer such as Pluronic, though any polymeric, or polymer-like, material that is compatible with native mammalian tissue can be used to generate the sacrificial wall. In some embodiments, the sacrificial wall is created using a bioink comprising a hydrophilic, non-ionic copolymer surfactant such as Pluronic F-127 (CAS No. 9003-11-6). In other embodiments, the sacrificial wall comprises one or more suitable biocompatible hydrogel polymers including, but not limited to, gelatin, agarose, alginate, polyvinyl alcohol (PVA), polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), paraffin wax, polystyrene (PS), saccharose (sucrose), dextran, pluronic F127, methylcellulose, calcium alginate, magnesium oxide (MgO), composite gels, fugitive inks, sacrificial carbohydrate glasses, silk fibroin, collagen, or combinations of any of the foregoing.

[0082] In other embodiments, the sacrificial wall is not deposited via a bioink, but is instead removable and made of a solid material such as metal, plastic, or other material. In this embodiment, the sacrificial wall is manually placed onto the permeable membrane and removed therefrom at the appropriate time and may be reusable, given proper cleaning.

[0083] In another embodiment, the layer-on-layer model lacks a sacrificial wall. In such embodiments, the foregoing step is omitted and the method of generating a layer-on-layer lung model proceeds with the attachment of a support structure and optional generation of a basal layer. Support Structure and Basal Layer

[0084] After (optionally) coating the permeable membrane with ECM proteins and (optionally) depositing a sacrificial wall onto the permeable membrane, the layer-on-layer lung model proceeds with attachment of a support structure to the bottom and / or side walls of the permeable membrane insert, oriented toward the side of the permeable membrane opposite that on which the lung stem cells are to be seeded (see, e.g., Figures 2, 3). In some embodiments, the support structure is a polymeric (e.g., plastic) 3D printed, biocompatible clip that is clipped, or press-fitted, onto the bottom of the permeable membrane insert, such that friction keeps the clip attached securely. In other embodiments, the support structure is a metal component that is 3D metal printed, machined, or cast to fit the permeable membrane insert, assuming tolerances are maintained. In still other embodiments, the support structure can be attached to the membrane insert by more conventional ways, for example glue, screws, adhesives, and the like. The support structure provides structural integrity to the lung model, allowing manipulability during and after culturing as may be needed. Given that role, the support structure may be made of any suitable, biologically inert material, for example plastic, metal, or other suitable materials, and may be fabricated using methods including additive or subtractive manufacturing.

[0085] The support structure also serves to support a basal layer, which is also added to the bottom of the lung model, on the side of the permeable membrane opposite that on which lung stem cells will be seeded (see, e.g., Figures 2, 3). The basal layer serves to support the remainder of the lung model and can be added to the model by rotating the permeable membrane insert, exposing the bottom surface of the permeable membrane, and creating the basal layer directly on the underside of the membrane.

[0086] In some embodiments, the support structure may be omitted. In such embodiments, the basal layer typically adheres to the bottom of the permeable membrane insert, becoming more permanently affixed thereto after polymerization (see below). In such embodiments, the composition of the basal layer is such that it displays adherent properties with respect to the membrane.

[0087] In various embodiments, the basal layer is a gel that is deposited (e.g., pipetted, bioprinted, etc.) onto the exposed bottom surface of the permeable membrane. In some embodiments, the basal layer comprises a liquid bioink mixed with endothelial cells (e.g., human pulmonary microvascular endothelial cells) and fibroblast cells that is pipetted onto the bottom surface of the permeable membrane insert to form a three-dimensional basal layer. In such embodiments, the liquid bioink is selected from collagen type I, gelatin methacrylate, collagen type I, collagen type IV, solubilized extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA), solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (e.g., Matrigel, Corning Incorporated, Glendale, AZ, USA), or combinations of the foregoing.

[0088] The amount of liquid bioink used to generate the basal layer can vary from about 50 μL – about 500 μL, in some embodiments about 100 μL, about 150 μL, about 200 μL, about 250 μL, about 300 μL, about 350 μL, about 400 μL, about 450 μL, or about 500 μL. The total volume of the basal layer can thus also vary from about 1 μL – about 500 μL, in some embodiments 1 μL, 5 μL, 10 μL, 25 μL, 50 μL, 75 μL, 100 μL, 250 μL, or 500 μL.

[0089] After deposition of a basal layer, the nascent lung model is polymerized. Polymerization may occur via any suitable means, for example in some embodiments polymerization occurs by incubating the models in a humidified incubator for a period of time sufficient to achieve complete, or nearly complete, polymerization of the sacrificial wall, the basal layer, and / or both. Suitable incubation / polymerization conditions can include, for example, temperatures of about 30 ºC – about 40 ºC (or about 30 ºC to about 37 ºC, or 37 ºC in other embodiments), at about 3% – about 8% CO2 (5% CO2 in one embodiment), and at a humidity level of about 90% – about 95% in a thermal chamber, such as a humidified incubator, for about 30 – about 90 minutes. The amount of time can vary. In some embodiments, polymerization occurs from 0.1 - 4 hours, in some embodiments 10 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours.

[0090] In other embodiments, the tissue may be polymerized with other suitable methods, including but not limited to non-thermal methods such as photopolymerization, chemical crosslinking, enzymatic crosslinking, self-assembly, interfacial polymerization, and / or combinations of any of the foregoing.

[0091] In some embodiments, the layer-on-layer model does not have a support structure or a basal layer, or both. In such embodiments, the foregoing step is omitted to the extent appropriate (i.e., by eliminating the addition of the support structure and / or the basal layer to the lung model) and the method of generating a layer-on-layer lung model proceeds with seeding the appropriate cells and the initiation of culture. Initiation

[0092] After coating the membrane, depositing the sacrificial wall, and creating the support and basal layers, the model culture is initiated. Initiation comprises the seeding of alveolar stem cells on the inner region of the permeable membrane insert and bronchial stem cells on the outer region of the same surface (see, e.g., Figures 2, 3). Seeding preferably occurs on the side of the permeable membrane opposite that on which the support structure and / or basal layer is / are added.

[0093] Initial seeding involves the creation of stem cell suspensions, which are then seeded onto the nascent, polymerized model as noted above. Generally, the stem cells comprise alveolar stem cells and bronchial cells. In various embodiments, stem cell suspensions can be made by resuspending bronchial cells and day 4 alveolar organoids in initiation medium (defined herein) in preparation for seeding. In one embodiment, about 500,000 bronchial cells are resuspended in approximately 60 μL of initiation medium and about a 50 μL dome of day 4 alveolar organoids is separately resuspended in about 20 μL of initiation medium in preparation for seeding.

[0094] The seeded density of the stem cell suspensions can vary, depending on the desired application for the lung model. In some embodiments, alveolar cells or dissociated alveolar organoids may be used, with alveolar stem cells seeded at a density of about 500,000 cells per model. In some embodiments, the cell density of bronchial cells is approximately 8,333 cells / µL, while single alveolar stem cells have a density of about 25,000 cells / μL. In some embodiments, the alveolar stem cells and bronchial stem cells are each independently present at cell densities ranging from about 1,000 cells / µL – about 50,000 cells / µL, in some embodiments about 1,000 cells / µL, about 2,000 cells / µL, about 3,000 cells / µL, about 5,000 cells / µL, about 8,000 cells / µL, about 13,000 cells / µL, about 21,000 cells / µL, about 34,000 cells / µL, or about 50,000 cells / µL. In some embodiments, the alveolar stem cells and bronchial stem cells are each independently present in the lung model at a range of about 1,000 – about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells.

[0095] Upon culture initiation (i.e., the seeding of stem cells onto the permeable membrane as noted above), the sacrificial wall either dissolves through interaction with the initiation medium or is mechanically removed. Initiation then proceeds initially for about 12 – about 36 hours, in some embodiments for about 24-hours, to ensure proper seeding of the stem cells on the lung model. After this initial seeding period, more initiation medium is added and the lung model is cultured, allowing the stem cells to proliferate atop the permeable membrane. In various embodiments, the proliferating cells form a monolayer, covering the entire membrane surface onto which they were seeded, including the void left by the wall (if any), creating a bronchoalveolar interface (see, e.g., Figure 6a).

[0096] During this initiation culturing time, initiation medium is continuously administered to the lung model until the microtissues reach a state suitable for transition to the maturation phase (e.g., until such time as a completely confluent monolayer of cells has grown across the surface of the membrane). The duration of culture time required for this initiation phase can therefore vary. In some embodiments, initiation culture time lasts from about 1 – about 7 days, concluding once the permeable membrane insert is fully confluent. The initiation phase may also be extended beyond 7 days to accommodate specific experimental conditions or cell types.

[0097] Culturing during the initiation phase involves submerged culture, where both sides of the lung model are fully covered by initiation medium. The volume of initiation medium added will vary based on the suitable configuration of insert – well combination that is selected (see above). Generally speaking, it is desirable to add a sufficient volume of initiation medium to each side of the lung model to ensure that submerged culture conditions exist. The volume of initiation medium can thus vary from about 0.1 mL to about 10L, in some embodiments 0.1 mL, 1 mL, 100 mL, 1L, 2L, 5L, or 10L. In those embodiments in which well plates are used in connection with a permeable membrane insert (see, e.g., Figure 4), a volume (e.g., about 0.5 mL) of initiation medium is added to the top of the monolayer and another volume (e.g., about 1.0 mL) is added to the bottom compartment of the well plate, below the permeable membrane insert.

[0098] In various embodiments, the initiation phase uses a specialized initiation medium that comprises 5 % serum, agonists for Wnt / β-catenin, EGFR, FGFR, and VEGFR signaling pathways, and antagonists for GSK3, BMP, p38 MAPK, ROCK, and TGFβ signaling pathways (see, e.g., Example 2). The medium can also comprise Y27632, a ROCK inhibitor, to ensure the viability and proliferation of lung epithelial stem cells, and ascorbic acid, to promote collagen deposition.

[0099] In some embodiments, the initiation media comprises VEGF-165, FGF-2, FGF-7 (10 ng / mL), FGF-10 (50 ng / mL), and EGF (5 ng / mL). In embodiments, the initiation media comprises 10 µM Y27632 (TargetMol, T1870). In some embodiments, the initiation media comprises CHIR-99021, LDN-193189, A-83-01, and / or SB202190. In some embodiments, CHIR-99021 (1.5 µM), a GSK3 inhibitor, is used to ensure stem cell proliferation and maintenance (Cayman, 13122). In some embodiments, LDN-193189 hydrochloride (100 nM), a BMP inhibitor, is added because it enhances the self-renewal of stem cells, preventing differentiation (PeproTech, 1066208). In some embodiments, A-83-01 (0.5 µM), an inhibitor of the TGFβ pathway (Cayman, 9001799), is included to maintain pluripotency by blocking differentiation signals. In some embodiments, SB202190, an inhibitor for p38k, which enhances the proliferation and survival of stem cells, is added at 250 nM (Targetmol, 2301). The initiation media ensures the establishment of the culture and tissue proliferation while also delaying premature differentiation.

[0100] In another embodiment, Noggin may be used in place of LDN-193189 hydrochloride.

[0101] The exact composition of the initiation medium may vary. Fetal bovine serum, when used, may be in a range from 0.1 to 10 % including but not limited to 0.1 %, 2.5 %, 5 %, 7.5 %, or 10 %. VEGF-165, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-2, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-7, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-10, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. EGF, when used, may be in a range from 0.1 to 50 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Y27632, when used, may be in a range from 0.1 to 30 µM, including but not limited to 0.1µM, 5 µM, 10 µM, 20 µM, or 30 µM. CHIR-99021, when used, may be in a range from 0.1 - 10 µM including but not limited to 0.1µM, 1 µM, 2 µM, 5 µM, or 10 µM. LDN-193189 hydrochloride, when used, may be in a range from 0.1 - 500 nM including but not limited to 0.1 nM, 50 nM, 100 nM, 200 nM, or 500 nM. A-83-01, when used, may be in a range from 0.1 nM to 1 µM including but not limited to 0.1 nM, 50 nM, 500 nM, or 1 µM. Ascorbic acid, when used, may be in a range from 0.05 mg / ml to 10 mg / ml, including but not limited to 0.05 mg / ml, 0.1mg / ml, 1mg / ml, 2,5 mg / ml, or 10 mg / ml.

[0102] In another embodiment, Noggin may be used in place of LDN-193189 hydrochloride.

[0103] In another embodiment, the initiation medium may lack VEGF-165 and FGF-2.

[0104] In another embodiment, other growth factors or cytokines may be included in the initiation medium to enhance specific aspects of tissue development. For example, including factors such as EGF, TGF-β, or HGF could influence differentiation pathways and improve the functional characteristics of the microtissues.

[0105] In some embodiments, the initiation medium is a combination of human lung organoid (hLO) expansion media and HFL1 media, both as presented in Example 2. In such embodiments, hLO and HFL1 media are mixed together in a 1:1 ratio, optionally supplementing with about 30 ng / mL of VEGF-165 and about 35 ng / mL of FGF-2.

[0106] In numerous embodiments, initiation is completed when the stem cells reach full confluence across the permeable membrane. Maturation

[0107] Thereafter, the maturation phase for the layer-on-layer model begins. The maturation phase is characterized by the creation of an air-liquid interface (ALI) culture, which allows for the diffusion of nutrients and media from the liquid into the tissue. In an ALI culture, the upper tissue surface is exposed to the air, while the rest of the lung model remains in contact with a maturation medium (defined herein). In that regard, the initiation medium is first removed from both sides of the lung model and is replaced, on one side only, with maturation medium.

[0108] In various embodiments, then, creating an air-liquid interface is achieved by removing medium from the top of the permeable membrane insert (i.e., from the side of the membrane containing now confluent stem cells). Air naturally displaces the volume once occupied by the initiation medium, exposing the apical membrane. Thereafter, enough maturation medium is added to submerge the basal layer, leaving the epithelium exposed to air. The amount of maturation medium needed to achieve this will vary depending on the size and shape of the insert at issue. In some embodiments, the volume of maturation medium used to submerge the basal layers is from about 0.1 mL to about 10L, in some embodiments about 0.1 mL, about 1 mL, about 100 mL, about 1L, about 2L, about 5L, or about 10L.

[0109] In some embodiments, varying the air-liquid interface conditions, such as adjusting the humidity or oxygen levels, may be employed to mimic different physiological or pathological states, providing a more versatile model. For example, the humidity and oxygen levels along the ALI can be adjusted to mimic the environment inside of a healthy mammalian lung, or to mimic a desired disease state.

[0110] In various embodiments, the maturation medium comprises about 10 % serum and agonists for FGFR (Fibroblast Growth Factor Receptor) and VEGFR (Vascular Endothelial Growth Factor Receptor) signaling pathways. Agonists targeting FGFR and VEGFR promote cellular growth and differentiation pathways within the lung model’s microtissues. In other embodiments, the maturation medium lacks VEGF-165 and FGF-2, which could be relevant for certain experimental setups or specific types of tissue cultures. Ascorbic acid, when used, may be in a range from 0.05 mg / ml to 10 mg / ml, including but not limited to 0.05 mg / ml, 0.1mg / ml, 1mg / ml, 2,5 mg / ml, or 10 mg / ml.

[0111] In some embodiments, the maturation medium is HFL1 media, as presented in Example 2, supplemented with about 30 ng / mL of both VEGF-165 and FGF-2.

[0112] The combination of maturation medium and air-liquid interface culture promotes tissue maturation by mimicking conditions found in a mature lung. In various embodiments, the lung model microtissues can be considered mature after 21 days of ALI culture in maturation medium, though the amount of time can vary depending on the application for which the lung model is being generated. In some embodiments, maturation can be determined by the observation of various markers in the cultured cells, such as the presence of differentiated cell types, ciliary beating, mucus production, surfactant production, cell confluency, and / or others, to name a few. Immunofluorescent staining may also be carried out to verify the quantity and presence of differentiated lung cell types.

[0113] Generally speaking, the culture of the lung models in the maturation phase typically occurs for at least about 21 days to attain the desired level of maturity. This extended culture duration allows for the comprehensive differentiation and maturation of the cells within the microtissues, leading to the formation of functional structures resembling aspects of native lung tissue. In some embodiments, the achievement of maturity is evaluated through the observation of ciliary beating - a characteristic motion of ciliated cells indicative of functional integrity. In some embodiments, the achievement of maturity is evaluated through immunostaining techniques that assess the presence and distribution of specific cellular markers indicative of lung functionality. Maturation of the microtissues may also be verified with the use of immunological fluorescent staining, targeting the differentiated cells and corresponding markers including but not limited to ciliated (acetylated-ɑ- tubulin+), goblet (Mucin 5AC+), club (club cell 10-kDa protein, CC10+), and cytokeratin 5 (Krt5+) bronchial stem cells. The methods of maturation observance used can vary. In some embodiments various markers, stains, events, and morphology may be used, among other measures, to assess the development and functionality of the microtissues. Combinations of any two or more techniques may also be used to demonstrate a desired level of microtissue maturity.

[0114] Once the microtissue derived from the stem cells reaches a desired level of maturity, the basal layer tends to exhibit branching vasculature, akin to vasculature found in the human lung. In those embodiments in which human pulmonary microvascular endothelial cells are used to generate a basal layer added to the bottom side of the permeable membrane, those endothelial cells self-pattern into vessels that serve to transport nutrients, remove waste and deliver oxygen to the lung model. In turn, this self-patterning recapitulates the structures that respiratory viruses leverage to infiltrate their hosts. These cells also push differentiation in the lung. In some embodiments, the presence of this type of microvasculature in the layer-on-layer model ensures that it is infectable with a mechanism of action similar to that of human lungs, while also preventing necrosis of the tissue allowing ever larger tissue constructs to be made, making the layer-on-layer model suitable for use as a model system in lung and / or lung-associated diseases.

[0115] In some embodiments, the microtissues of the layer-on-layer lung model exhibit ciliary beating. In some embodiments, the microtissues of the layer-on-layer lung model exhibit production of mucus. In some embodiments, the microtissues of the layer-on-layer lung model exhibit both ciliary beating and production of mucus. Cells

[0116] In some embodiments, the layer-on-layer model contains only one lung epithelial cell type.

[0117] In some embodiments, the layer-on-layer model lacks fibroblasts and / or endothelial cells.

[0118] In some embodiments, differentiated lung stem cells may be used in place of lung epithelial stem cells.

[0119] In some embodiments, the lung models may contain either primary stem cells or iPSC-derived cells, or a combination thereof.

[0120] In some embodiments, other cells can be added to the lung models, barring lung epithelial stem cells, for example fibroblasts, microvascular endothelial cells, and / or smooth muscle cells can be added in order to generate more specialized lung models.

[0121] To maintain consistent cell counts in the microtissues, in some embodiments a standardized number of cells can be defined for a specific lung model, with each individual lung model type having its own standardized cell count. To effectuate this, cells can be manufactured and cryopreserved at specific concentrations, where they can be used to streamline the later bioprocess, for example by serving as the cells for resuspension in the initiation phase. In some embodiments, a standardized number of cells reflecting one or more of the ratios of cells found in the lung can be generated, manufactured, and cryopreserved in order to efficiently produce lung microtissues in high numbers.

[0122] In an embodiment, stem cells may be present in the layer-on-layer model at cell densities ranging from about 1,000 cells / µL – about 50,000 cells / µL, in some embodiments about 1,000 cells / µL, about 2,000 cells / µL, about 3,000 cells / µL, about 5,000 cells / µL, about 8,000 cells / µL, about 13,000 cells / µL, about 21,000 cells / µL, about 34,000 cells / µL, or about 50,000 cells / µL.

[0123] In an embodiment, non-stem cells may be present in the layer-on-layer model at cell densities ranging from about 1,000 cells / µL – about 50,000 cells / µL, in some embodiments about 1,000 cells / µL, about 2,000 cells / µL, about 3,000 cells / µL, about 5,000 cells / µL, about 8,000 cells / µL, about 13,000 cells / µL, about 21,000 cells / µL, about 34,000 cells / µL, or about 50,000 cells / µL.

[0124] The amount of cells per each cell type in each model may vary. In some embodiments each cell type in an individual model can vary from about 1 - about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells.

[0125] In some embodiments, cell types in a layer-on-layer model’s bioink is present in a ratio of alveolar epithelial: endothelial: interstitial: macrophages: smooth muscle: bronchial: other of 65-70:25-35:15-25:10-20:0.1-10:5-15:0.1-2, in some embodiments the ratio is 68:30:20:15:5:10:1. In some embodiments the layer-on-layer model recapitulates other layered tissues or organs, including, but not limited to, the skin, intestine, liver, kidney, pancreas, stomach, bladder, testes, ovaries, bone marrow, bone, teeth, retina, brain cortex, blood-brain barrier, thymus, heart, spleen, blood vessels, fat, cartilage, muscle, tendons and ligaments. In these embodiments, tissue specific cells, progenitors or stem cells may be included in the layer- on-layer model. 3D Branching Distal Lung Model

[0126] The branched model is an engineered tissue designed to recapitulate the in vivo structures found in the human distal lung. Grown with stem cells, endothelial cells, and mesenchymal cells, the model provides an accurate representation of the distal lung’s architecture and is useful for a wide range of research, therapeutic, and / or diagnostic applications. Broadly speaking, a mesenchymal hydrogel bath is created by mixing endothelial cells and fibroblasts. Lung epithelial stem cells and smooth muscle cells are deposited into the bath along with various growth factors, where lung tissue self-patterns. Thereafter an air-liquid interface is created as described herein, helping to mature the tissue and provide a route of entry to the apical membrane.

[0127] This 3D branching lung model can be used for a wide variety of applications, including viral pathophysiology studies, drug discovery, personalized therapy, and others. Additionally, a 3D branching lung model may be used to perform lung grafts to restore lung function in patients, for example who may be experiencing lung dysfunction or failure.3D branching lung models may be used in patients that require a lung transplant as well, creating a supply of engineered lung tissue.

[0128] The 3D branching distal lung model replicates the native architecture of terminal bronchioles and air sacs, facilitating the formation of bronchoalveolar junctions and supporting cellular differentiation. Preferably, this model can be achieved via precise deposition of bronchial cells and alveolar organoid fragments within a mesenchyme- containing hydrogel bath, such deposition being facilitated by advanced bioprinting hardware and optimized bioprocessing techniques.

[0129] In various embodiments, construction of the 3D branching distal lung model includes the deposition of bronchial cells and alveolar organoid fragments within a specialized hydrogel matrix, fostering self-assembly and morphogenesis akin to native lung tissue. The incorporation of a structural hydrogel ensures structural integrity and cellular functionality within the model.

[0130] In various embodiments, generation of a 3D branching lung model includes endothelial cell seeding, supporting hydrogel infill, and sequential bioprinting of bronchial and alveolar cell populations, culminating in model maturation and differentiation under controlled culture conditions.

[0131] Additionally, the methods describing the fabrication of the 3D branching lung model are also applicable to the fabrication of other tissues and organs composed of repeating functional units or with layered, hierarchical, or branched structures. One non-limiting embodiment is the structure of a branched blood vessel where fibroblasts, smooth muscle cells, endothelial cells, or their associated progenitors or stem cells, are deposited within the hydrogel bath. Another, non-limiting embodiment is the structure of the intestine where the hydrogel bath includes mesenchymal and vascular cells and intestinal mucosal cells, progenitors or stem cells are deposited within it.

[0132] One non-limiting embodiment of a method of producing a 3D branching lung model is as follows. Permeable membrane

[0133] In various embodiments, fabrication of a 3D branched lung model begins with the seeding of a permeable membrane that will be used for air-liquid interfaces with pulmonary microvascular endothelial cells. The permeable membrane can be of any configuration that allows for the seeding of such cells. For example, the membrane may be a “stand-alone” component or it may be integrated into a support structure that is suitable for insertion into a tissue culture well. The permeability of the membrane can vary, to suit the needs of the lung model, and may range from a pore size of 0.4 μm – 8.0 μm (e.g., Falcon and / or Transwell Permeable Supports, Corning Incorporated, Glendale, AZ, USA).

[0134] In some embodiments, such as that depicted in Figure 10a, the 3D branching model uses a well plate and compatible permeable membrane insert. In such embodiments, the permeable membrane insert sits on top of a standardized 6-well plate, with each insert extending into an individual well (Figure 10a), though any suitable culture vessel may be used, for example 6-well permeable membrane inserts and plates, 12-well permeable membrane inserts and plates, 24-well permeable membrane inserts and plates, 48-well permeable membrane inserts and plates, bioreactors, industrial vats, and so on. The configuration of the permeable insert – well combination is not particularly limited and may be adjusted to suit the desired size of the lung model.

[0135] Seeding initially involves the creation of cell suspensions, which are then seeded onto the permeable membrane. Generally, endothelial cells, such as pulmonary microvascular endothelial cells, are used for seeding and are grown to confluence along the entire surface of the membrane. In various embodiments, stem cell suspensions can be made by resuspending endothelial cells in a growth medium (i.e., DMEM or the like) in preparation for seeding. The volume of growth medium utilized for the cell suspensions can vary to suit an individual lung model. In some embodiments, the amount of growth media used for a cell suspension is from about 0.1 mL - about 100L, in some embodiments about 0.1 mL, about 1 mL, about 100 mL, about 1L , about 2L, about 5L, about 10L, or about 100L. In one embodiment, 6 permeable membrane inserts are seeded, each with a total of approximately 500,000 pulmonary microvascular endothelial cells resuspended in about 1.5 mL of growth media, 0.5 mL of which is seeded onto the top compartment of the insert and 1.0 mL on the bottom. The seeded membrane is then cultured until the permeable membrane is confluent.

[0136] The seeded density of the endothelial cell suspensions can vary, depending on the desired application for the lung model. In some embodiments, pulmonary microvascular endothelial cells may be present in the branching model at a cell density ranging from about 1,000 cells / µL - about 1,000,000 cells / µL, in some embodiments about 1,000 cells / µL, about 5,000 cells / µL, about 20,000 cells / µL, about 50,000 cells / µL, about 100,000 cells / µL, about 250,000 cells / µL, about 500,000 cells / µL, about 750,000 cells / µL, or about 1,000,000 cells / µL. In some embodiments, the endothelial cells are present in the lung model at a range of about 1 – about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells. Mesenchymal Bulk Hydrogel

[0137] Once the permeable membrane reaches confluence, a mesenchymal bulk hydrogel comprising mesenchymal cells and additional endothelial cells is made. The mesenchymal bulk hydrogel serves as structural support for the lung model microtissue, similar to connective tissue found in the human body. It has the dual purpose of supporting the microtissue during the printing stages, and protecting and encapsulating it during and after maturation. The fibroblasts and endothelial cells contained within it perform structural support, nutrient transport, and signaling functions, enabling the tissue to proliferate and differentiate.

[0138] Generally, the bulk hydrogel is prepared by encapsulating (i.e., resuspending) mesenchymal cells and endothelial cells in a hydrogel containing additional components such as laminin, collagen, entactin / nidogen, and heparin sulfate proteoglycans, which aid in the formation of bulk tissue. In some embodiments, the hydrogel also comprises bovine collagen type I (e.g., at 5 mg / mL, Advanced Biomatrix, Carlsbad, CA, USA) and a reduced growth factor basement membrane extract (e.g., at 8 mg / mL, (e.g., Cultrex BME, Bio- Techne, Minneapolis, MN, USA). The ratio of collagen : basement membrane extract can vary. In some embodiments, the ratio is 40% collagen : 60 % extract. In other embodiments, the ratio of collagen : extract is selected from 1:4, 1:1, 2:3, and 1:2. The hydrogel itself may be made of any suitable hydrogel polymeric matrix, including but not limited to gelatin methacrylate, fibrin, sodium alginate, collagen type I, PLGA, collagen type IV, solubilized extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio- Techne, Minneapolis, MN, USA), solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (e.g., Matrigel, Corning Incorporated, Glendale, AZ, USA), or combinations of the foregoing.

[0139] Following the resuspension of endothelial cells and mesenchymal cells into this bulk mesenchymal hydrogel, any media present from the endothelial seeding of the membrane is removed and the bulk hydrogel is then placed into contact with the now confluent permeable membrane (see, e.g., Figure 10a). The amount of bulk hydrogel placed into contact with the membrane can vary, for example from 1 μL – 1000 μL, in some embodiments 1 μL, 5 μL, 10 μL, 25 μL, 50 μL, 75 μL, 100 μL, 250 μL, 500 μL, or 1000 μL. In some embodiments, approximately 300 μL of the bulk hydrogel comprising approximately 500,000 fibroblast cells and approximately 500,000 pulmonary microvascular cells is placed into contact with the confluent membrane.

[0140] In some embodiments, each cell type may individually be present in a 3D branching model at this point of bioprocessing at a cell density of about 1,000 cells / µL – about 1,000,000 cells / µL, in some embodiments about 1,000 cells / µL, about 5,000 cells / µL, about 20,000 cells / µL, about 50,000 cells / µL, about 100,000 cells / µL, about 250,000 cells / µL, about 500,000 cells / µL, about 750,000 cells / µL, or about 1,000,000 cells / µL. The total number of cells present in the 3D lung model at this point in production can vary. In some embodiments, the total number of cells present is from about 1 – about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells.

[0141] Once the bulk hydrogel cells are placed into contact with the confluent permeable membrane, the nascent 3D lung model is typically ready for bioprinting (see, e.g., Figure 10a). If bioprinting will not occur immediately, then the endothelial seeded model can be kept at about 2 – about 8 º C until the lung epithelial stem cells are deposited into the hydrogel. Lung Epithelial Stem Cells / Bioink

[0142] At this point in processing, the bioink(s) necessary to deposit alveolar and / or bronchial stem cells onto the confluent membrane are generated. In various embodiments, the bioink(s) is a hydrogel that comprises bovine collagen type I (e.g., at 5 mg / mL, Advanced Biomatrix, Carlsbad, CA, USA) and a reduced growth factor basement membrane extract (e.g., at 8 mg / mL, (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA), into which one or more types of cells are inoculated. The ratio of collagen : basement membrane extract in the bioink(s) can vary. In some embodiments, the ratio is 40% collagen : 60 % extract. In other embodiments, the ratio of collagen : extract is selected from 1:4, 1:1, 2:3, and 1:2. The hydrogel itself may be made of any suitable hydrogel polymeric matrix, including but not limited to gelatin methacrylate, fibrin, sodium alginate, collagen type I, PLGA, collagen type IV, solubilized extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA), solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (e.g., Matrigel, Corning Incorporated, Glendale, AZ, USA), or combinations of the foregoing.

[0143] In some embodiments, two lung stem cell bioinks are prepared prior to deposition into the hydrogel: an alveolar bioink, which in some embodiments may be an alveolar organoid bioink, and a bronchial stem cell bioink. The alveolar bioink can be made by creating a suspension of alveolar cells, whole alveolar organoids, fragments of alveolar organoids, or any combination thereof in a bioink hydrogel. In one embodiment, about 6 domes of 50 μL / day 4 whole alveolar organoids and / or organoid fragments is suspended in 30 μL of bioink hydrogel. Similarly, the bronchial bioink can be created by creating a suspension of bronchial stem cells and / or bronchial smooth muscle cells in a bioink hydrogel. In one embodiment, a bronchial bioink is prepared by suspending about 6 million bronchial stem cells and about 3 million bronchial smooth muscle cells in 30 μL of bioink hydrogel.

[0144] In some embodiments, each cell type may individually be present in the bioinks at a cell density of about 1,000 cells / µL - about 1,000,000 cells / µL, in some embodiments about 1,000 cells / µL, about 5,000 cells / µL, about 20,000 cells / µL, about 50,000 cells / µL, about 100,000 cells / µL, about 250,000 cells / µL, about 500,000 cells / µL, about 750,000 cells / µL, or about 1,000,000 cells / µL. In some embodiments, the amount each cell type in a bioink may vary from about 1 - about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells.

[0145] Once the bioinks are prepared and the bulk hydrogel cells are placed into contact with the confluent permeable membrane, bioprinting can begin (see, e.g., Figure 10a). Bioprinting

[0146] Following the creation of the bioinks, the deposition of cells and / or organoids occurs. In various embodiments, deposition of 3D structures occurs directly onto the confluent membrane. This may be accomplished in any number of ways, for example the permeable membrane (and its insert, if applicable) can be placed into the deposition device, where the bioinks are deposited through the mesenchyme + hydrogel suspension (see above and, e.g., Figure 10a) and directly onto the membrane. Deposition of the bioinks can occur in a number of ways. For example, a bioink may be deposited manually via pipetting, hand- based extrusion, or use of a microneedle and micromanipulator. Typically, however, deposition of the bioinks into the membrane occurs via a bioprinting device, such as a bioprinter (e.g., a computer-controlled bioprinter / deposition system, for example an Inkredible+ bioprinter, Cellink, Carlsbad, CA, USA). After deposition, a polymerization step can occur in order to retain the positioning of the bioinks originally laid down by the deposition system. In various embodiments, bioinks are deposited such that relevant structures form during the culture period, for example bioinks can be deposited such that alveolar air sacs form and fuse with bronchioles to create a distal lung structure.

[0147] In some embodiments, a solid piece (i.e., a stamp) may be used as an alternative to bioprinting. In these embodiments, there step of generating bioinks can be skipped, in favor of placing a single, stamped construct onto the confluent membrane. The stamp may be placed in the bulk hydrogel (see, e.g., Figure 10a) prior to polymerization (as described herein) to cast a shape into the bulk hydrogel. The resulting void is then filled with cells that form into a lung tissue. In some embodiments, an alveolar bioink is deposited first, followed by a bronchial bioink. In other embodiments, a bronchial bioink is deposited first, followed by an alveolar bioink. In some embodiments, alveolar bioinks are placed as dots (small volumes extruded at a point without motion of the deposition nozzle), or as a line. Then, bronchial cells are placed adjacently or in contact with the printed alveolar bioink as a branching line structure mimicking the distal lung.

[0148] In some embodiments, the deposition device is a coaxial printhead used to print two cell types and bioinks at the same time. In these embodiments, growth medium is in the inner needle and lung epithelial cells are in the outer needle to aid in lumen formation. In similar embodiments, lung epithelial cells are in the inner needle and a combination of endothelial cells and fibroblast cells are in the outer needle to aid in formation of microvasculature close in proximity to the lung epithelia.

[0149] In other embodiments, the deposition device is a triaxial printhead used to print three cell types, or three distinct bioinks, at one time. In these embodiments, the innermost needle contains a growth medium, the middle needle contains lung epithelial cells, and the outer needle contains a combination of endothelial cells and fibroblast cells. In similar embodiments, lung epithelial cells are in the innermost needle, a combination of smooth muscle cells, endothelial cells and fibroblasts are in the middle needle, and a combination of endothelial cells and fibroblasts are in the outer needle. These embodiments aid in the formation of a continuous lumen, while also promoting formation of microvasculature surrounding the lung epithelia.

[0150] In still other embodiments, the deposition device is a quadaxial printhead used to print four cell types, or four distinct bioinks, at one time. In these embodiments, the innermost needle contains growth medium, the second innermost needle contains lung epithelial cells, the third innermost needle contains smooth muscle cells, and the outermost needle contains a combination of endothelial cells with fibroblasts. These embodiments aid in the formation of a continuous lumen, while also promoting formation of microvasculature surrounding the lung epithelia.

[0151] In still other embodiments, the deposition device is a quadaxial printhead

[0152] Once deposition is complete, the tissue is polymerized as described herein and then cultured. In embodiments, the tissue may be polymerized for about 0.1 – about 4 hours, in some embodiments about 10 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, or about 4 hours.

[0153] In still other embodiments, the bioprinting processes in the present disclosure may include cells that form into other tissues composed of repeating functional units or with layered, hierarchical, or branched structures. These tissues include, but are not limited to, the intestine, liver, kidney, pancreas, thymus, lymphatic system, testes, ovaries, bone marrow, blood-brain barrier, heart, spleen, blood vessels, fat, cartilage, muscle, tendons and ligaments. In these embodiments, tissue specific cells, progenitors or stem cells may be included in the 3D branching model. Initiation

[0154] The culture regime of the 3D branching model is split between an initiation phase and a maturation phase (see, e.g., Figure 10b). The initiation phase uses an initiation medium (defined herein). The initiation medium ensures the establishment of the culture and tissue proliferation while also delaying premature differentiation. In various embodiments, the initiation medium comprises 5 % serum, agonists for Wnt / β-catenin, EGFR, FGFR, and VEGFR signaling pathways, and antagonists for GSK3, BMP, p38 MAPK, ROCK, and TGFβ signaling pathways (see, e.g., Example 2). The medium can also comprise Y27632, a ROCK inhibitor, to ensure the viability and proliferation of lung epithelial stem cells. Ascorbic acid can be added to the medium to promote collagen deposition for improved structural integrity of the tissues.

[0155] In some embodiments, the initiation media comprises VEGF-165, FGF-2, FGF-7 (10 ng / mL), FGF-10 (50 ng / mL), and EGF (5 ng / mL). In embodiments, the initiation media comprises 10 µM Y27632 (TargetMol, T1870). In some embodiments, the initiation media comprises CHIR-99021, LDN-193189, A-83-01, and / or SB202190. In some embodiments, CHIR-99021 (1.5 µM), a GSK3 inhibitor, is used to ensure stem cell proliferation and maintenance (Cayman, 13122). In some embodiments, LDN-193189 hydrochloride (100 nM), a BMP inhibitor, is added because it enhances the self-renewal of stem cells, preventing differentiation (PeproTech, 1066208). In some embodiments, A-83-01 (0.5 µM), an inhibitor of the TGFβ pathway (Cayman, 9001799), is included to maintain pluripotency by blocking differentiation signals. In some embodiments, SB202190, an inhibitor for p38k, which enhances the proliferation and survival of stem cells, is added at 250 nM (Targetmol, 2301). The initiation media ensures the establishment of the culture and tissue proliferation while also delaying premature differentiation.

[0156] In another embodiment, Noggin may be used in place of LDN-193189 hydrochloride.

[0157] The exact composition of the initiation medium may vary. Fetal bovine serum, when used, may be in a range from 0.1 to 10 % including but not limited to 0.1 %, 2.5 %, 5 %, 7.5 %, or 10 %. VEGF-165, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-2, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-7, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-10, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. EGF, when used, may be in a range from 0.1 to 50 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Y27632, when used, may be in a range from 0.1 to 30 µM, including but not limited to 0.1µM, 5 µM, 10 µM, 20 µM, or 30 µM. CHIR-99021, when used, may be in a range from 0.1 - 10 µM including but not limited to 0.1µM, 1 µM, 2 µM, 5 µM, or 10 µM. LDN-193189 hydrochloride, when used, may be in a range from 0.1 - 500 nM including but not limited to 0.1 nM, 50 nM, 100 nM, 200 nM, or 500 nM. A-83-01, when used, may be in a range from 0.1 nM to 1 µM including but not limited to 0.1 nM, 50 nM, 500 nM, or 1 µM. Ascorbic acid, when used, may be in a range from 0.05 mg / ml to 10 mg / ml, including but not limited to 0.05 mg / ml, 0.1mg / ml, 1mg / ml, 2,5 mg / ml, or 10 mg / ml.

[0158] In another embodiment, Noggin may be used in place of LDN-193189 hydrochloride.

[0159] In another embodiment, the initiation medium may lack VEGF-165 and FGF-2.

[0160] In another embodiment, other growth factors or cytokines may be included in the initiation medium to enhance specific aspects of tissue development. For example, including factors such as EGF, TGF-β, or HGF could influence differentiation pathways and improve the functional characteristics of the microtissues.

[0161] In some embodiments, the initiation medium is a combination of human lung organoid (hLO) expansion media and HFL1 media, both as presented in Example 2. In such embodiments, hLO and HFL1 media are mixed together in a 1:1 ratio, optionally supplementing with about 30 ng / mL of VEGF-165 and about 35 ng / mL of FGF-2.

[0162] During the initiation phase of culture, the model is completely submerged in the initiation medium. In some embodiments, initiation medium is added to both the top and bottom of the culture, ensuring contact with all sides of the model. In embodiments such as those depicted in Figure 10a, about 0.5 mL of initiation medium is added to the top of the tissue, and about 1.0 mL is added to the bottom. The volume of initiation medium used to ensure submerged culture can vary to suit the bioprocessing setup. For example, the volume of initiation medium can vary from about 0.1 mL to about 10L, in some embodiments about 0.1 mL, about 1 mL, about 100 mL, about 1L, about 2L, about 5L, or about 10L.

[0163] This initiation phase typically lasts about 3 to about 5 days, concluding once the 3D lung structures form cohesive and connected structures. In some embodiments, the initiation phase can last from 1 to 7 days, in some embodiments beyond 7 days to accommodate specific experimental conditions or cell types. In the embodiment depicted in Figure 10b, the initiation phase of culture lasts for 5 days.

[0164] During the initiation phase of culture, the bronchial stem cells and bronchial smooth muscle cells self-pattern to form tubes with lumina, similar to the in vivo lung. Similarly, alveolar organoids fuse to form large sacs. Over time, the bronchioles and alveolar sacs fuse to form a holistic distal lung structure. Initiation medium is regularly added during this process, preserving the stem cell populations in both epithelial cell types.

[0165] Once the 3D lung structures of the lung model form cohesive and connected structures, the maturation phase of culture can begin. Maturation

[0166] The maturation phase of culture is characterized by the creation of an air-liquid interface. In this setup, one side of the tissue surface is exposed to air, and the other side is in contact with a maturation medium (defined herein). The combination of maturation medium and air-liquid interface culture promotes tissue maturation by mimicking conditions found in the mature human lung. In that regard, the initiation medium is first removed from both sides of the lung model and is replaced, on one side only, with maturation medium.

[0167] In several embodiments, the maturation medium comprises 10 % serum and agonists for FGFR and VEGFR signaling pathways. Enough maturation medium is added to submerge the basal layer, leaving the epithelium exposed to air. The amount of maturation medium needed to achieve this will vary depending on the size and shape of the insert at issue. In some embodiments, about 0.75 mL of maturation medium is added to the bottom of the tissue, though this amount can vary from about 0.1 mL to about 10L, in some embodiments about 0.1 mL, about 1 mL, about 100 mL, about 1L, about 2L, about 5L, or about 10L.

[0168] In various embodiments, the maturation medium comprises about 10 % serum and agonists for FGFR (Fibroblast Growth Factor Receptor) and VEGFR(Vascular Endothelial Growth Factor Receptor) signaling pathways. Agonists targeting FGFR and VEGFR promote cellular growth and differentiation pathways within the lung model’s microtissues. In other embodiments, maturation medium lacks VEGF-165 and FGF-2, which could be relevant for certain experimental setups or specific types of tissue cultures. In other embodiments, the maturation media further comprises alternative growth factors and / or cytokines to enhance specific aspects of tissue development. For example, factors such as EGF, TGF-β, or HGF may be added to influence differentiation pathways and improve the functional characteristics of the lung model’s microtissues.

[0169] In some embodiments, the maturation medium is HFL1 media, as presented in Example 2, supplemented with about 30 ng / mL of both VEGF-165 and FGF-2.

[0170] The air-liquid interface facilitates maturation of a 3D branching lung model by mimicking conditions found in the mature human lung. There are numerous ways to create an air-liquid interface. In some embodiments, an air-liquid interface may be created using a computer-controlled deposition device to deposit bronchial cells (e.g., via deposition of a bronchial stem cell bioink) along the z-axis inside the mesenchymal bulk hydrogel. The deposition continues until the cell-laden bioink has exited the top surface of the hydrogel. As the cells form bronchial tubes, the center of the tube is excavated, where a liquid filled lumen forms. Liquid is then removed, for example by vacuum suction, opening the 3D branching model to the air.

[0171] In other embodiments, the inner nozzle of a coaxial printhead is filled with a sacrificial hydrogel, and the outer nozzle is filled with lung stem cells mixed with bioink. These two bioinks are simultaneously deposited into the bulk mesenchymal hydrogel, forming lung tissue with a sacrificial interior. After printing, the tissue is cultured, and the sacrificial material is removed either by dilution with medium, pneumatic removal using vacuum suction, alternative methods, or combinations thereof. In some embodiments, medium is added to the top of the tissue surface where it diffuses into the tissue, dissolving the sacrificial material.

[0172] In still other embodiments, liquid inside the bronchial regions of the 3D lung model may be removed via vacuum suction, liquid may be from the lung model by inverting the model and allowing liquid to flow out of the bronchial microtissue, the tissue may be separated using a sterile scalpel, and / or a needle can be attached to the bronchial region before printing. Removal of liquid by inversion may be aided by the application of mechanical force while the tissue is inverted, the deformation of which will displace the liquid from the bronchial region, creating a hollow lung tissue that is open to the air. When a needle is attached to the bronchial region before printing, liquid can be removed from the lung model and air can be pumped in and out of the lung tissue, via the needle.

[0173] In some embodiments, varying the air-liquid interface conditions during the maturation phase of culture, such as adjusting the humidity or oxygen levels, may be employed to mimic different physiological or pathological states, providing a more versatile model. For example, the humidity and oxygen levels along the ALI can be adjusted to mimic the environment inside of a healthy mammalian lung, or to mimic a desired disease state.

[0174] In various embodiments, the lung model microtissues can be considered mature after 21 days of ALI culture in maturation medium, though the amount of time can vary depending on the application for which the lung model is being generated. In some embodiments, maturation can be determined by the observation of various markers in the cultured cells, such as the presence of differentiated cell types, ciliary beating, mucus production, cell confluency, and / or others, to name a few. Immunofluorescent staining may also be carried out to verify the quantity and presence of differentiated lung cell types. Overall, maturation of both the bronchial and alveolar compartments in 3D branching lung models can be evidenced by the presence of mature, differentiated cell endotypes and the presence of expected morphology, such as ciliary beating.

[0175] The methods of maturation observance used can vary. In some embodiments various markers, stains, events, and morphology may be used, among other measures, to assess the development and functionality of the microtissues. Combinations of any two or more techniques may also be used to demonstrate a desired level of microtissue maturity.

[0176] Generally speaking, the culture of the lung microtissue models in the maturation phase typically occurs for at least about 21 days to attain the desired level of maturity. This culture duration allows for the comprehensive differentiation and maturation of the cells within the microtissues, leading to the formation of functional structures resembling aspects of native lung tissue. In some embodiments, the achievement of maturity is evaluated through the observation of ciliary beating - a characteristic motion of ciliated cells indicative of functional integrity. In some embodiments, the achievement of maturity is evaluated through immunostaining techniques that assess the presence and distribution of specific cellular markers indicative of lung functionality. Maturation of micro tissues may also be verified with the use of immunological fluorescent staining, targeting the differentiated cells and corresponding markers including but not limited to ciliated (acetylated-ɑ-tubulin+), goblet (Mucin 5AC+), club (club cell 10-kDa protein, CC10+), and cytokeratin 5 (Krt5+) bronchial stem cells.

[0177] For the 3D branching model, maturity of the microtissues can be observed in additional ways. For example, observation of apical-in polarity can be evidenced by enrichment of Filamentous actin (F-actin, via phalloidin staining) towards the bronchial lumen and the proximal localization of differentiated cell types in the lumen. Close inspection of the tissues confirming the presence of pseudostratified morphology with luminal facing ciliated, goblet, and club cells can indicate microtissue maturity. Bronchoalveolar junctions / interfaces, or a situation where two distinct populations of epithelial cells are present, can be observed and the presence of balloon-like projections expressing Human type I cell 56-kDa protein (HT1-56, alveolar type 1 marker) with branching structures protruding towards these alveolar air sacs that are Krt5+ suggests that these are bronchial cells. A bronchoalveolar junction-like feature is typically formed after maturation of the bioprinted tissues.

[0178] The presence of tight junctions in both the epithelial and endothelial tissues can also be indicative of maturity. Zonula Occludens-1 (ZO-1), a tight junction marker, can be used in this 3D model and in the layer-on-layer model (above) to observe that both tissue layers express the tight junction marker. The formation of tight junctions suggests that both tissue layers exhibit maturation features necessary for barrier function. the formation of tight junctions is also relevant for the subsequent study of viral infection, as such insults dissociate and destabilize the tight junctions. Regarding alveolar type 1 (HT1-56) and type 2 (Anti-Prosurfactant Protein C, abbreviated as Pro-SPC), lung models produced according to the methods provided herein confirm that the majority of the airsac-like lining is HT1- 56+ with some interspersed cuboidal cells expressing Pro-SPC – this morphology is a typical feature of the lung alveolus.

[0179] In various embodiments, the mesenchymal bulk hydrogel will contain branching vasculature. Like the layer-on-layer model, the endothelial cells self-pattern into vessels that serve to transport nutrients to, remove waste from, and deliver oxygen to the microtissues, while also recapitulating the structures that respiratory viruses leverage to infiltrate their hosts. Additionally, the vasculature aids in the differentiation of lung epithelial stem cells, ensuring physiological accuracy. In viral pathophysiological studies, it is desirable for a proper air-liquid interface to be present, which can be facilitated by depositing cells such that the cells form tubes open to ambient air, as provided herein.

[0180] Following differentiation, the 3D branching lung model is ready for various applications. Cells

[0181] In some embodiments, each cell type may individually be present in the 3D branching lung model at a cell density of about 1,000 cells / µL - about 1,000,000 cells / µL, in some embodiments about 1,000 cells / µL, about 5,000 cells / µL, about 20,000 cells / µL, about 50,000 cells / µL, about 100,000 cells / µL, about 250,000 cells / µL, about 500,000 cells / µL, about 750,000 cells / µL, or about 1,000,000 cells / µL. In some embodiments, the amount each cell type in a lung model may vary from about 1 - about 11,000,000,000 cells, in some embodiments about 1,000 cells, about 100,000 cells, about 1,000,000 cells, about 1,000,000,000 cells, about 2,000,000,000 cells, about 3,000,000,000 cells, about 4,000,000,000 cells, about 5,000,000,000 cells, about 6,000,000,000 cells, about 7,000,000,000 cells, about 8,000,000,000 cells, about 9,000,000,000 cells, about 10,000,000,000 cells, or about 11,000,000,000 cells.

[0182] In some embodiments, cell types in a 3D branching lung model’s bioink is present in a ratio of alveolar epithelial: endothelial: interstitial: macrophages: smooth muscle: bronchial: other of 65-70:25-35:15-25:10-20:0.1-10:5-15:0.1-2, in some embodiments the ratio is 68:30:20:15:5:10:1.

[0183] In various embodiments, any one or more cell type described herein may be excluded from use in a particular model.

[0184] In other embodiments, some or all of the cell types found in the human lung may be present in lung microtissues including but not limited to Club Cells, Ciliated Cells, Basal Cells, Goblet Cells, Mucous Cells, Serous Cells, Ionocytes, Neuroendocrine Cells, Tuft Cells, Alveolar Epithelial Type 1 Cells, Alveolar Epithelial Type 2 Cells, Artery Cells, Vein Cells, Capillary Cells, Bronchial Vessel cells, Lymphatic Cells, Vascular Smooth Muscles, Airway Smooth Muscles, Fibroblasts, Myofibroblasts, Lipofibroblasts, Pericytes, Mesothelial Cells, Intrinsic Neurons, Glial Cells, B Cells, Plasma Cells, CD8+ Mem / Eff T Cells, CD8+ Naive T Cells, CD4+ Mem / Eff Cells, CD4+ Naive T Cells, Natural Killer Cells, Natural Killer T Cells, Neutrophils, Basophils, Mast Cells, Eosinophils, Megakaryocytes, Macrophages, Plasmacytoid Dendritic Cells, Myeloid Dendritic Cells 1, Myeloid Dendritic Cells 2, Classical Monocytes, Intermediate Monocytes, Nonclassical Monocytes, and combinations thereof.

[0185] In some embodiments, the 3D branching model may lack alveolar organoids. In such embodiments, alveolar sacs form from the bronchial structure. In other embodiments, differentiated lung stem cells may be deposited in place of lung epithelial stem cells. In still other embodiments, alveolar cells may be used in lieu of alveolar organoid fragments. In still other embodiments, whole alveolar organoids may be used in lieu of alveolar organoid fragments.

[0186] In some embodiments, the 3D branching lung model exhibits ciliary beating. In some embodiments, the 3D branching model exhibits production of mucus. In some embodiments, the 3D branching model exhibits ciliary beating and production of mucus

[0187] In some embodiments the 3D branching model recapitulates tissues other than the lung that are composed of repeating functional units or with layered, hierarchical, or branched structures. These tissues include, but are not limited to, the intestine, liver, kidney, pancreas, thymus, lymphatic system, testes, ovaries, bone marrow, blood-brain barrier, heart, spleen, blood vessels, fat, cartilage, muscle, tendons and ligaments. In these embodiments, tissue specific cells, progenitors or stem cells may be included in the 3D branching model. Large-Scale Manufacture of Lung Stem Cells

[0188] In one aspect, the present disclosure provides methods for the large scale production of lung epithelial stem cells. The disclosed methods have the potential to revolutionize their manufacturing process by significantly reducing costs, lowering the required technical expertise, and enabling large-scale production.

[0189] Generally, the methods comprise coating cell culture vessels or bioreactors with extracellular matrix proteins and feeding undifferentiated lung stem cells a tailored medium. The combination of these two elements allows lung epithelial cells to proliferate while remaining immature. This culture method can reduce the cost of upscaling lung epithelial stem cells by about twenty times in comparison to traditional 3D culture methods. Additionally, the disclosed method represents an improvement over known 2D culture methods in both quantity and quality of cells harvested. Traditional methods of 2D culture are insufficient in upscaling lung epithelial cells, because the cells reach senescence by passage 2-3, and can often prematurely terminally differentiate. In sharp contrast, the disclosed method of lung epithelial stem culture is capable of preserving the immature morphology into passages in excess of passage 8, passage 9, passage 10, and beyond. The matrix protein coating in combination with the proprietary lung epithelial stem cell medium allows for the large-scale manufacturing of lung (e.g., alveolar and bronchial) stem cells by mimicking the microenvironment necessary for lung stem cells to grow.

[0190] In the disclosed methods, cell culture vessels, bioreactors, and / or the like are first coated with extracellular matrix (ECM) proteins. The exact composition of the extracellular matrix proteins can vary, though in some embodiments the composition of the extracellular matrix proteins is similar to solubilized extracellular matrix proteins derived from a basement membrane (e.g., Cultrex BME, Bio-Techne, Minneapolis, MN, USA). In various embodiments, the extracellular matrix proteins are selected from laminin, collagen, entactin / nidogen, heparan sulfate proteoglycans, and combinations of any of the foregoing. The proteins are dissolved in a suitable solvent, such as tissue culture media (e.g., DMEM medium, Basal medium, or the like), initiation medium (described herein), or other suitable liquid. The concentration of each extracellular matrix protein in the suitable solvent can vary. In some embodiments, the concentration of each ECM protein in solution is independently about 0.1 % – about 25 %. In some embodiments, the total concentration of all ECM proteins in the suitable solvent is 1 %, 2 %, 5 %, 10 %, 15 %, 20 %, or 25 %.

[0191] This extracellular matrix protein solution is placed into contact with those surfaces of the cell culture vessels, bioreactors, and / or the like that are to be coated for a period of time sufficient for the extracellular matrix proteins to adhere to the surfaces, for example from about 1 minute to about 2 days’ time. The duration of the coating process can vary and includes times such as about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 60 minutes, about 3 hours, about 1 day, or about 2 days. After the coating time, the medium could either be removed, or the cells could be directly seeded onto the coated vessels. In some embodiments, excess coating solution is removed, for example by aspiration or other suitable methods, leaving the coated vessel.

[0192] Thereafter, lung stem cells are cultured in the coated vessel(s) in an initiation medium (defined herein). Lung stem cells can be sourced from a donor, commercially (e.g., Sciencell Research Laboratories, Carlsbad, CA, USA), from primary cells or induced pluripotent stem cells (iPSCs), or from any appropriate source. In some embodiments, the lung stem cells comprise alveolar epithelial cells, bronchial epithelial cells, or both. In some embodiments, prior to initiation of culture of the lung stem cells, the multipotent stem cells are isolated from the contaminating cell population by using the human lung organoid expansion (hLO) medium described in Example 2.

[0193] After isolation, the multipotent stem cells, which comprises both alveolar and bronchial epithelial stem cells, are cultured in initiation medium on the coated tissue culture vessels (created as per the above). In various embodiments, the initiation medium comprises 5 % serum, agonists for Wnt / β-catenin, EGFR, FGFR, and VEGFR signaling pathways, and antagonists for GSK3, BMP, p38 MAPK, ROCK, and TGFβ signaling pathways (see, e.g., Example 2). The medium can also comprise Y27632, a ROCK inhibitor, to ensure the viability and proliferation of lung stem cells. The initiation media ensures the establishment of the culture and tissue proliferation while also delaying premature differentiation.

[0194] In some embodiments, the initiation media comprises VEGF-165, FGF-2, FGF-7 (10 ng / mL), FGF-10 (50 ng / mL), and EGF (5 ng / mL). In embodiments, the initiation media comprises 10 µM Y27632 (TargetMol, T1870). In some embodiments, the initiation media comprises CHIR-99021, LDN-193189, A-83-01, and / or SB202190. In some embodiments, CHIR-99021 (1.5 µM), a GSK3 inhibitor, is used to ensure stem cell proliferation and maintenance (Cayman, 13122). In some embodiments, LDN-193189 hydrochloride (100 nM), a BMP inhibitor, is added because it enhances the self-renewal of stem cells, preventing differentiation (PeproTech, 1066208). In some embodiments, A-83-01 (0.5 µM), an inhibitor of the TGFβ pathway (Cayman, 9001799), is included to maintain pluripotency by blocking differentiation signals. In some embodiments, SB202190, an inhibitor for p38k, which enhances the proliferation and survival of stem cells, is added at 250 nM (Targetmol, 2301).

[0195] In another embodiment, Noggin may be used in place of LDN-193189 hydrochloride.

[0196] The exact composition of the initiation medium may vary. Fetal bovine serum, when used, may be in a range from 0.1 to 10 % including but not limited to 0.1 %, 2.5 %, 5 %, 7.5 %, or 10 %. VEGF-165, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-2, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-7, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. FGF-10, when used, may be in a range from 0.1 to 100 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, or 100 ng / mL. EGF, when used, may be in a range from 0.1 to 50 ng / mL, including but not limited to 0.1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL. Y27632, when used, may be in a range from 0.1 to 30 µM, including but not limited to 0.1µM, 5 µM, 10 µM, 20 µM, or 30 µM. CHIR-99021, when used, may be in a range from 0.1 - 10 µM including but not limited to 0.1µM, 1 µM, 2 µM, 5 µM, or 10 µM. LDN-193189 hydrochloride, when used, may be in a range from 0.1 - 500 nM including but not limited to 0.1 nM, 50 nM, 100 nM, 200 nM, or 500 nM. A-83-01, when used, may be in a range from 0.1 nM to 1 µM including but not limited to 0.1 nM, 50 nM, 500 nM, or 1 µM. Ascorbic acid, when used, may be in a range from 0.05 mg / ml to 10 mg / ml, including but not limited to 0.05 mg / ml, 0.1mg / ml, 1mg / ml, 2,5 mg / ml, or 10 mg / ml.

[0197] In some embodiments, the initiation medium may lack VEGF-165 and FGF-2.

[0198] In some embodiments, other growth factors or cytokines may be included in the initiation medium to enhance specific aspects of tissue development. For example, including factors such as EGF, TGF-β, or HGF could influence differentiation pathways and potentially prolong the time in which maturation is delayed.

[0199] In some embodiments, the initiation medium is a combination of human lung organoid (hLO) expansion media and HFL1 media, both as presented in Example 2. In such embodiments, hLO and HFL1 media are mixed together in a 1:1 ratio, optionally supplementing with about 30 ng / mL of VEGF-165 and about 35 ng / mL of FGF-2.

[0200] In other embodiments, serum free initiation medium can be used.

[0201] In all of the foregoing embodiments, the disclosed method allows lung stem cells to proliferate, while remaining immature, for at least 8 passages, in some embodiments 9 passages, in some embodiments 10 passages, in some embodiments 11 passages, in some embodiments 12 passages, in some embodiments 13 passages, in some embodiments 14 passages, in some embodiments 15 passages, in some embodiments 16 passages, in some embodiments 17 passages, in some embodiments 18 passages, in some embodiments 19 passages, in some embodiments 20 passages, and in some embodiments greater than 20 passages. Uses

[0202] Applications of the lung microtissues are wide-ranging. For instance, lung models provided by the present disclosure can be used: as a model system to study the effects of viral infections, for implantation in patients to replace or improve function of diseased lungs, for drug discovery, disease modeling, synthetic biology, drug screening, biodefense studies, developmental studies, and the like.

[0203] With respect to their use as models of viral infections, Example 6 provides data indicating that lung microtissues made according to the present disclosure are useful for the study of viral pathogenesis and host response.

[0204] Given the data provided in Example 6, it is reasonable to conclude that different respiratory viruses may also be used to infect both the layer-on-layer model and the branching model, and that the results will be similarly successful. This will allow for a broader study of viral pathogenesis and host responses to various viral pathogens.

[0205] In some embodiments, 3D branching lung models provided by the present disclosure can be cut open, exposing the apical membrane and thereby facilitating viral infection. This method ensures direct access of the virus to the cells and can mimic different infection scenarios, demonstrating the great flexibility of the disclosed lung models as strong exemplary systems.

[0206] In those embodiments of the 3D branching lung models that utilize a stamp to create a void, the air-exposed tissue that results can be exposed to a virus, thereby facilitating viral infection. This technique can help in studying the effects of air exposure on viral infectivity and tissue response.

[0207] As noted above, some embodiments of the 3D branching lung models involve printing the cells of the branching construct in a way such that the tubular bronchial formation forms a pathway from the bronchial lumen to the top surface of the hydrogel bath. This results in an opening to the inner lumen of the structure which can be used to deposit viral particles for viral infection studies, replicating a natural route of infection, and enhancing the physiological relevance of the study.

[0208] Additionally, in other embodiments, the virus can be replaced with a drug and / or biological therapeutic, transforming the lung models into tools for drug discovery. This approach allows for the evaluation of drug efficacy and safety in a controlled in vitro environment, providing valuable insights for pharmaceutical development.

[0209] Additional examples of suitable uses are shown in the following hypothetical scenarios:

[0210] Case 1: A patient with lung cancer is in poor health and needs a tailored drug concoction. a. Application: The patient’s cells are harvested and reprogrammed as iPSCs. They are then differentiated into endothelial cells, lung epithelial stem cells, fibroblasts, and bronchial smooth muscle cells. Lung models are made in accordance with this disclosure, creating a personalized distal lung or lung tissue. This tissue is then subjected to various drugs until the optimal treatment is found for the patient, saving the patient and healthcare provider time.

[0211] Case 2: A patient needs a lung transplant, but cannot source a viable lung tissue due to shortage. a. Application: The patient’s cells are harvested and reprogrammed as iPSCs. They are then differentiated into endothelial cells, lung epithelial stem cells, fibroblasts, and bronchial smooth muscle cells. Lung models are made in accordance with this disclosure, creating a personalized whole lung tissue. The lung tissue is grown until maturity and implanted into the patient.

[0212] Case 3: A novel respiratory virus emerges, data regarding the virus is limited. a. Application: Lung tissues are prepared in accordance with the disclosure. The novel virus is exposed to the lung tissues, allowing researchers to observe the virus and its associated pathophysiology with greater accuracy relative to single cell cultures and organoid cultures.

[0213] Overall, the layer-on-layer lung models and the 3D branching lung models represent significant advancements in tissue engineering, offering researchers unprecedented capabilities to study complex pulmonary biology and investigate disease mechanisms in vitro. With potential applications in drug discovery, disease modeling, and therapeutic development, this innovation holds promise for revolutionizing the field of pulmonary research.

[0214] In one embodiment, the lung models may be grown into artificial lungs and implanted into an animal or human patient.

[0215] In other embodiments, the lung models may be used to investigate the effects of disease pathology and progression.

[0216] In other embodiments, the lung models may be used for personalized medicine. Lungs may be taken from the patient to create bespoke lung-microtissues for the purposes of testing drugs on a person’s specific lung biology, enabling increased efficacy in treatment.

[0217] In other embodiments, the lung model may incorporate immune cells including but not limited to alveolar macrophages.

[0218] In other embodiments, the 3D branching lung model may be the size of the human lung after printing, and grown / matured to form an engineered lung suitable for implantation.

[0219] In other embodiments, lung grafts may be made by placing the lung model in a patient’s lung, restoring natural lung function.

[0220] In other embodiments, a drug assay may be created using the lung models.

[0221] The materials and methods provided in the following examples are for illustrative purposes only and are not intended to be limiting. EXAMPLES Example 1 - Generation and Maintenance of Lung Stem Cells

[0222] Human-derived lung stem cells were sourced from Sciencell both human-derived alveolar epithelial cells (3200) and human-derived bronchial epithelial cells (3210). Multipotent stem cells were selected from the contaminating cells using in-house human lung organoid expansion (hLO) medium. Once multipotent stem cells were isolated, both alveolar and bronchial epithelial stem cells were cultured on treated petri dishes coated with a 5% Cultrex solution. The Cultrex was dissolved into a coating medium, DMEM:F12 medium. The cells were directly seeded upon the petri dishes following a coating time of 30 minutes. The coating medium was not removed prior to seeding. Five hundred thousand stem cells of each type were separately seeded onto a 10 cm petri dish. As the cells were multiplied, the number and size of vessels required to manufacture the cells increased, resulting in a total yield of about 750 million cells. Lung epithelial stem cells were cultured with initiation medium, detailed in Example 2. Example 1A - 2D CULTURE AND EXPANSION OF LUNG MICROVASCULAR ENDOTHELIAL CELL LINE (HPMEC), PRIMARY LUNG BRONCHIAL SMOOTH MUSCLE CELL LINE (HBSMC), AND LUNG STROMAL CELL LINES HFL1 AND HULEC5a

[0223] The following protocol may be used for all cell types noted in the title. Corresponding complete growth medium should be used for each cell type as follows:

[0224] HPMEC: Endothelial cell media MV2 Promocell, C22121

[0225] HBSMC: Smooth Muscle Cell Medium, Sciencell, 1101 HFL1:

[0002] HULEC5a:

[0226] Initially, cryopreserved cells are thawed. Set the water bath to 37°C, and then spray the culture hood with 70% ethanol to disinfect it. Vials are to be removed from liquid nitrogen storage, sprayed with ethanol, and submerged in the water bath below the cap for approximately two minutes, ensuring the ice is almost completely melted. The vials are then brought into the culture hood, and the cell contents shall be transferred to a 15 mL conical vial using a pipette. The cryovial is then rinsed with complete growth media, and the contents are to be combined with the initial transfer to the conical vial, which is then topped off to 10 mL with growth media. The suspension should be centrifuged at 600xg for 5 minutes at 4°C, and the supernatant is then discarded. The cell pellet is to be resuspended in 10 mL of culture media, and cells are then plated into a 10 cm tissue culture dish and incubated at 37°C with 5% CO2. Cell attachment is to be checked after 24 hours, and the confluency monitored daily until it reaches 80%, typically within 2-5 days.

[0227] For passaging and upscaling, once the cells reach 80% confluency and are confirmed healthy without signs of contamination, the biosafety cabinet is sterilized with 70% ethanol. The culture plates are also sterilized with ethanol, and the media is to be aspirated. Cells are washed with 5 mL of PBS, which is then aspirated. Trypsin or TrypLE is to be added to the cells and incubated at 37°C for 3-5 minutes to dislodge the cells, confirm detachment under a microscope. Any remaining attached cells are to be incubated for an additional 2 minutes, if necessary. Do not exceed a total of 15 minutes. Trypsin is deactivated by adding 7.5 mL of complete growth media, and cell clumps are to be broken up into single cells by pipetting up and down. The cells are then centrifuged at 600xg for 5 minutes at 4°C. Aspirate the supernatant, and the cell pellet shall be resuspended in 10 mL of media. Cells are plated at a density of 8,000 to 12,000 cells per cm² or a split ratio between 1:2 and 1:6, depending on the desired scale. The dishes are incubated at 37°C and checked after 24 hours for cell attachment. The cultures reach confluence within 3-5 days.

[0228] For cryopreservation and banking, cells at 80% confluency are used. The biosafety cabinet and culture plates are sterilized with 70% ethanol. Media is to be aspirated away, and cells shall be washed with 5 mL of PBS, which is then aspirated. Trypsin or TrypLE is added, and cells are incubated at 37°C for 5-10 minutes to dislodge them. After confirming cell detachment, complete growth media is added to deactivate the Trypsin, and cell clumps are broken into single cells. Cells are then centrifuged at 600xg for 5 minutes at 4°C. The cell pellet is resuspended in freezing media, prepared by combining complete growth medium, FBS, and DMSO. The cells are aliquoted into prechilled, prelabeled cryovials at a density of 0.5 - 3.0 x 10⁶ cells per vial. The cryovials are then placed in a Mr. Frosty freezing container prefilled with isopropyl alcohol and prechilled. The container is stored in a -80°C freezer for at least 5 hours before transferring the vials to a liquid nitrogen tank for long- term storage. Example 2 - Media Formulations

[0229] Lung microtissues are cultivated using a dual-media approach consisting of initiation medium and maturation medium. Both initiation and maturation media formulations incorporate agonists targeting VEGFR (Vascular Endothelial Growth Factor Receptor) and FGFR (Fibroblast Growth Factor Receptor), which promote cellular growth and differentiation pathways within the microtissues. The initiation medium is enriched with Y27632, a specific inhibitor of Rho-associated protein kinase (ROCK), which enhances the proliferation and viability of stem cells during the initial stages of culture.

[0230] Human lung organoid expansion (hLO) medium is a cell culture medium intended to grow alveolar epithelial stem cells in the form of 3D organoids. It is a medium containing agonists for Wnt / β-catenin, EGFR, FGFR, VEGFR signaling and antagonists for GSK3, BMP, p38 MAPK, ROCK, and TGFβ signaling pathways. It is formulated with the combination of the following reagents at the specified concentrations in Advanced DMEM / F12 (Millipore Sigma, SCM162): HEPES (10 mM), GlutaMax (L-Alanyl L- glutamine) (1X, 10 mM), Antibiotic-Antimycotic(1X), N2 (1X), B27 minus vit A (1X), N- Acetyl cysteine (1.25 mM), Nicotinamide (5 mM), R-spondin conditioned media (10%), EGF (Epidermal Growth Factor) (10 ng / mL), FGF2 (Fibroblast Growth Factor 2) (5 ng / mL), FGF7 (Fibroblast Growth Factor 7) (20 ng / mL), FGF10 (Fibroblast Growth Factor 10) (100 ng / mL), LDN193189 (200 nM), SB202190 (500 nM), A8301 (1 µM), CHIR99021 (3 µM), Y27632 (10 µM,), Ascorbic Acid (0.05 mg / ml).

[0231] HFL1 medium is a medium intended to grow HFL1 cells, which are fibroblasts isolated from the lungs of a human embryo. Components are added to DMEM / F12 (Corning, 15-090-CV) at the following concentrations: DMEM / F12 (88%), FBS (10%), Alanyl-Glutamine (1X, 10 mM), Antibiotic-Antimycotic (1X).

[0232] Initiation medium is a combination of the previous two cell growth media: human lung organoid expansion (hLO) medium and HFL1 medium. Initiation medium is formulated with 5 % fetal bovine serum, VEGF-165, FGF-2, FGF-7 (10 ng / mL), FGF-10 (50 ng / mL) and EGF (5 ng / mL) are used. All growth factors were sourced from Pepro-Tech. The medium contains 15 µM Y27632 (TargetMol, T1870), a ROCK inhibitor that ensures the viability and proliferation of the lung epithelial stem cells. CHIR-99021 (3 µM), a GSK3 inhibitor, is used to ensure stem cell proliferation and maintenance (Cayman, 13122). LDN- 193189 hydrochloride (200 nM), a BMP inhibitor, enhances the self-renewal of stem cells, preventing differentiation (PeproTech, 1066208). A-83-01 (1 µM) is an inhibitor of the TGFβ pathway (Cayman, 9001799). Initiation medium was created by mixing one part of hLO medium with one part of HFL1 medium, supplementing with FGF-2 and VEGF-165 to the specified concentrations. It is also possible to further supplement 10 µM Y27632 for the first three days of culture.

[0233] Maturation medium is a 10% serum-supplemented medium made by combining HFL1 medium with VEGF-165 (30 ng / mL) and FGF-2 (30 ng / mL).

[0234] Both initiation and maturation media were supplemented with pro-angiogenic factors including VEGF-165 (30 ng / mL) and FGF-2 (35 ng / mL) to promote vasculogenesis and maturation. Table 1: Media Formulation Summary Table 2: Human Lung Organoid Expansion Medium Formulation Table 3: HFL1 Medium Formulation Table 4: HULEC Medium Formulation Example 3 - Workflow for the Layer-on-Layer Lung Model

[0235] A 12-well plate and compatible inserts were sourced from Celltreat (230621). First, a permeable membrane was coated with Cultrex dissolved in DMEM:F12 medium at a 5% concentration. Coating time is at least 30 minutes. The precise deposition of the sacrificial wall, made of pluronic, is conducted following the coating of the permeable membrane insert. The wall is deposited onto the upper surface of the permeable membrane insert with a bioprinter. The permeable membrane insert is placed into the bioprinter in a custom permeable membrane insert mount. The GCODE file containing commands to print the wall is loaded onto the printer and executed with the aid of an internet-connected bioprinter UI and computer. Following the deposition of the pluronic wall, a support structure is attached to the bottom of the permeable membrane insert. The basal layer is then added to the bottom of the permeable membrane insert by flipping the permeable membrane insert, exposing the bottom surface of the membrane. The basal layer comprises 100 µL of collagen type I (5mg / mL), sourced from Advanced Biomatrix (5074), containing endothelial and fibroblast cells. The basal layer is pipetted under the support structure onto the bottom surface of the membrane. The model contains approximately 500,000 endothelial and fibroblast cells each. The structure is then polymerized for 90 minutes, at 37 ºC, 5% CO2, and humidity of 90%– 95% in a humidified incubator. Subsequently, the model is removed from the incubator and the culture is initiated.

[0236] Initiation entails the seeding of alveolar stem cells on the inner region of the upper surface of the permeable membrane insert, and bronchial stem cells on the outer region of the same surface. Initial seeding is 500,000 bronchial cells per model resuspended in 60 μL of initiation medium, and one 50 μL dome of day 4 alveolar organoids in 20 μL of initiation medium per model. The cell density of bronchial cells is approximately 8,333 cells / μL. Following 24 hours of the seeding period and subsequent dissolution of the pluronic wall, the model is nourished with the addition of an initiation medium, as described in Example 2, for five to seven days until maturation. The tissue is fully submerged in the initiation medium during this period. Maturation medium is added after determining the confluence of the model. During maturation, an air-liquid interface is created by exposing the top of the tissue to air. Maturation medium is only added to the bottom compartment of the well in this phase. With the conclusion of the maturation phase, the monolayer differentiates to form an epithelium closely mimicking the one found in the human lung. Example 3A - 3D BIOPRINTING THE LAYER-ON-LAYER LUNG MODEL

[0237] Initially, all necessary instruments and materials, including the Flux1 V2 Bioprinter, various reagents, and cell lines, were prepared and assembled. The protocol began with the preparation of the bioprinting environment, which involved cleaning and sterilizing the bioprinter and associated equipment with 70% ethanol. The cells required for bioprinting were cryopreserved and stored at -80°C to maintain their viability. Specific media formulations were prepared in advance, including basal media and initiation media, which were supplemented with growth factors such as VEGF165 and FGF2, and stored at appropriate temperatures for use during the process.

[0238] Bioprinting commenced with the coating of transwell membranes using a Cultrex and DMEM solution. After aspirating the coating solution, the inserts were washed and dried to prepare them for cell seeding. The Pluronic bioink, which was essential for forming the structural support, was cooled and loaded into the bioprinter's extruder. The bioink was extruded to create a pluronic wall on the transwell inserts, which were then placed into a tissue culture plate for further processing.

[0239] For cell encapsulation, HFL1 and HPMEC cells were thawed and mixed with a collagen solution, which was then added to the bioprinted structures. The collagen layer was polymerized by placing the models in a humidified incubator.

[0240] Subsequent steps involved preparing the alveolar and bronchial epithelial cells. The alveolar organoids were thawed and resuspended in initiation media, while the bronchial epithelial cells were similarly thawed, washed, and resuspended. These cells were then carefully seeded into their respective compartments within the bioprinted models: alveolar cells in the inner compartment and bronchial cells in the outer compartment.

[0241] The cultured constructs were maintained under specific conditions to ensure their maturation. Initially, initiation media was added to both compartments of the wells. As the epithelial cells reached confluence around day 7, the media was switched to maturation media, and the constructs were cultured until day 21. Media changes were performed carefully to avoid disrupting the cell layers.

[0242] In cases where the collagen layer showed defects, additional collagen was added to rectify the structure. The process involved removing the media, inverting the models, and applying more collagen to restore the extracellular matrix. These steps were repeated as necessary until the collagen was adequately polymerized and the structural integrity of the model was restored. Example 3B - 3D BIOPRINTING THE LAYER-ON-LAYER LIVER MODEL

[0243] Initially, all necessary instruments and materials, including the Flux1 V2 Bioprinter, various reagents, and cell lines, are prepared and assembled. The protocol begins with the preparation of the bioprinting environment, which involves cleaning and sterilizing the bioprinter and associated equipment with 70% ethanol. The cells required for bioprinting are cryopreserved and stored at -80°C to maintain their viability. Specific media formulations are prepared in advance, including basal media and initiation media, which are supplemented with growth factors and stored at appropriate temperatures for use during the process.

[0244] Bioprinting commences with the coating of transwell membranes using a Cultrex and DMEM solution. After aspirating the coating solution, the inserts are washed and dried to prepare them for cell seeding. The Pluronic bioink, which is essential for forming the structural support, is cooled and loaded into the bioprinter's extruder. The bioink is extruded to create a pluronic wall on the transwell inserts, which are then placed into a tissue culture plate for further processing.

[0245] For cell encapsulation, vascular smooth muscle cells vascular endothelial cells are thawed and mixed with a collagen solution, which is then added to the bioprinted structures. The collagen layer is polymerized by placing the models in a humidified incubator.

[0246] Subsequent steps involve preparing the cholangiocytes and hepatocytes. The cholangiocytes are thawed and resuspended in media, while the hepatocytes are similarly thawed, washed, and resuspended. These cells are then carefully seeded into their respective compartments within the bioprinted models: cholangiocytes in the inner compartment and hepatocytes in the outer compartment.

[0247] The cultured constructs are maintained under specific conditions to ensure their maturation. Media changes are performed carefully to avoid disrupting the cell layers.

[0248] In cases where the collagen layer shows defects, additional collagen is added to rectify the structure. The process involves removing the media, inverting the models, and applying more collagen to restore the extracellular matrix. These steps are repeated as necessary until the collagen is adequately polymerized and the structural integrity of the model is restored. Example 4 - Workflow for the 3D Branching Lung Model

[0249] A 12-well plate and compatible inserts were sourced from Celltreat (230621). Fabrication of the branched models begins with the seeding of a permeable membrane insert with pulmonary endothelial cells. Approximately 500,000 pulmonary microvascular endothelial cells resuspended in 0.5 mL of growth medium are seeded atop the insert and 1.0 mL of growth medium is pipetted below the insert. Culture of the cells is continued until the permeable membrane is confluent. Following this, creation of the bulk tissue is fabricated with the encapsulation of mesenchymal cells and endothelial cells in collagen type I (5mg / mL) sourced from Advanced Biomatrix (5074), and Cultrex reduced growth factor basement membrane extract (8 mg / mL) from Bio Techne (3533-005-02). The collagen and Cultrex are mixed such that there is a 4:6 ratio of collagen to Cultrex. The bulk hydrogel and cells are then placed in the permeable membrane insert, and 300 μL of the mixture is placed into the insert. 500,000 human-derived fibroblast cells and 500,000 pulmonary microvascular cells are present in each permeable insert. The inserts are kept cold from 2 to 8 degrees Celsius until the lung epithelial stem cells are deposited into the hydrogel. Two lung stem cell bioinks are made prior to deposition into the hydrogel bath. An alveolar organoid bioink is made, and a bronchial stem cell bioink is made. The alveolar bioink is made by taking 6 domes of 50 μL / dome day 4 alveolar organoids and mixing them with 30 μL of acellular hydrogel matrix. A new container is used for the creation of bronchial bioink, which is made by taking 6 million bronchial stem cells and 3 million bronchial smooth muscle cells and adding them to an acellular hydrogel matrix. 30 μL is used to create the bronchial bioink.

[0250] Following the creation of the bioink, deposition of the cells and organoids is required. The alveolar organoids are deposited first, followed by the bronchial bioink. The permeable membrane insert is placed into the bioprinter in a custom permeable membrane insert mount. The GCODE file containing commands to print the alveolar print is loaded onto the printer and executed with the aid of an internet-connected bioprinter UI and computer. Next, the GCODE containing the commands to form the bronchial print are loaded onto the bioprinter and executed. Following the deposition of the bioinks into the mesenchymal hydrogel bath matrix, a polymerization step retains the positioning originally laid down by the bioprinter. The tissue is polymerized for 40 minutes after the deposition step, and cultured.

[0251] The first culture phase is a submerged culture, where the tissue is fully covered by initiation medium.0.5 mL of initiation medium is added to the top of the tissue, and 1.0 mL is added to the bottom of the tissue. This phase lasts about 5 days, concluding once the 3D lung structures form cohesive and connected structures. Thereafter, the maturation phase begins, the defining aspect of which being an air-liquid interface culture. In this culture, the tissue surface is exposed to the air, and a maturation medium is used comprising 10 % serum and agonists for signaling pathways FGFR and VEGFR. Enough maturation medium is added to submerge the basal layer, leaving the epithelium exposed to air. 0.75 mL of maturation medium is added to the bottom of the tissue. Tissues are considered mature after 21 days of culture in some but not all cases.

[0252] The bioprocessing workflow encompasses endothelial cell seeding, support hydrogel infill, and sequential bioprinting of bronchial and alveolar cell populations, culminating in model maturation and differentiation under controlled culture conditions. Example 4A - BIOPRINTING AND CULTURE OF 3D BRANCHING DISTAL LUNG MODEL

[0253] Initially, the necessary reagents were prepared. Cryopreserved cells were used for convenience, despite the benefits of using live cells for the overall health of the model. Basal media consisting of Advanced DMEM / F12 supplemented with Y27632 ROCK inhibitor was prepared. Cultrex, a key component for creating the support hydrogel, was thawed overnight at 4°C. Additional reagents included HFL1 media, hLO media, and a mixture of PureCol Ezgel and Cultrex to form the bulk hydrogel. This preparation was followed by the setup of the bioprinter and the cooling of the necessary syringes and needles at 4°C.

[0254] For the seeding of the endothelial layer, one vial of HPMEC cells was thawed and resuspended in DMEM / F12 media. After centrifugation, the cells were seeded onto permeable membrane inserts in a 12-well tissue culture plate and cultured until a confluency of at least 80% was achieved.

[0255] In the infill step, cells were mixed with the Cultrex bulk to achieve the desired concentration. This mixture was then used to fill permeable membrane inserts, which were subsequently stored at 4°C. The bronchial epithelial cells, smooth muscle cells, and alveolar organoids were prepared for bioprinting by thawing and resuspending them in the Cultrex bulk. These preparations were labeled accordingly for easy identification during the bioprinting process.

[0256] Bioprinting was carried out using a FLUX1 v2 bioprinter equipped with cooled positive displacement extruders. Example 4B - BIOPRINTING AND CULTURE OF INTESTINAL MODEL

[0257] Initially, the necessary reagents are prepared. Cryopreserved cells are used for convenience, despite the benefits of using live cells for the overall health of the model. Basal media is prepared. Cultrex, a key component for creating the support hydrogel, is thawed overnight at 4°C. Additional reagents include cell specific supplemented growth medium, and a mixture of PureCol Ezgel and Cultrex to form the bulk hydrogel. This preparation is followed by the setup of the bioprinter and the cooling of the necessary syringes and needles at 4°C.

[0258] For the seeding of the vascular endothelial cell layer, one vial of human intestinal microvascular endothelial cells is thawed and resuspended in specific media. After centrifugation, the cells are seeded onto permeable membrane inserts in a 12-well tissue culture plate and cultured until a confluency of at least 80% is achieved.

[0259] In the infill step, mesenchymal cells are mixed with the Cultrex bulk to achieve the desired concentration. This mixture is then used to fill permeable membrane inserts, which are subsequently stored at 4°C. The intestinal mucosal cells, progenitor cells and stem cells are prepared for bioprinting by thawing and resuspending them in the Cultrex bulk. These preparations are labeled accordingly for easy identification during the bioprinting process.

[0260] Bioprinting is carried out using a FLUX1 v2 bioprinter equipped with cooled positive displacement extruders. Example 4C - BIOPRINTING AND CULTURE OF LIVER MODEL

[0261] Initially, the necessary reagents are prepared. Cryopreserved cells are used for convenience, despite the benefits of using live cells for the overall health of the model. Basal media is prepared. Cultrex, a key component for creating the support hydrogel, is thawed overnight at 4°C. Additional reagents include cell specific supplemented growth medium, and a mixture of PureCol Ezgel and Cultrex to form the bulk hydrogel. This preparation is followed by the setup of the bioprinter and the cooling of the necessary syringes and needles at 4°C.

[0262] For the seeding of the vascular endothelial cell layer, one vial of human liver sinusoidal microvascular endothelial cells is thawed and resuspended in specific media. After centrifugation, the cells are seeded onto permeable membrane inserts in a 12-well tissue culture plate and cultured until a confluency of at least 80% is achieved.

[0263] In the infill step, mesenchymal cells are mixed with the Cultrex bulk to achieve the desired concentration. This mixture is then used to fill permeable membrane inserts, which are subsequently stored at 4°C. The hepatocytes, cholangiocyte, hepatic stellate cells, liver progenitor cells and stem cells are prepared for bioprinting by thawing and resuspending them in the Cultrex bulk. These preparations are labeled accordingly for easy identification during the bioprinting process.

[0264] Bioprinting is carried out using a FLUX1 v2 bioprinter equipped with cooled positive displacement extruders. Example 4D - BIOPRINTING AND CULTURE OF KIDNEY MODEL

[0265] Initially, the necessary reagents are prepared. Cryopreserved cells are used for convenience, despite the benefits of using live cells for the overall health of the model. Basal media is prepared. Cultrex, a key component for creating the support hydrogel, is thawed overnight at 4°C. Additional reagents include cell specific supplemented growth medium, and a mixture of PureCol Ezgel and Cultrex to form the bulk hydrogel. This preparation is followed by the setup of the bioprinter and the cooling of the necessary syringes and needles at 4°C.

[0266] For the seeding of the vascular endothelial cell layer, one vial of human glomerular microvascular endothelial cells is thawed and resuspended in specific media. After centrifugation, the cells are seeded onto permeable membrane inserts in a 12-well tissue culture plate and cultured until a confluency of at least 80% is achieved.

[0267] In the infill step, mesenchymal cells are mixed with the Cultrex bulk to achieve the desired concentration. This mixture is then used to fill permeable membrane inserts, which are subsequently stored at 4°C. The parietal epithelial cells, podocytes, proximal tubule epithelial cells, thin limb cells, thick ascending limb cells, macula densa cells, distal convoluted tubule cells, connecting tubule cells, collecting duct cells, transitional urothelial cells, peritubular capillary endothelial cells, progenitor cells and stem cells are prepared for bioprinting by thawing and resuspending them in the Cultrex bulk. These preparations are labeled accordingly for easy identification during the bioprinting process.

[0268] Bioprinting is carried out using a FLUX1 v2 bioprinter equipped with cooled positive displacement extruders. Example 5 - General Culture of Lung Microtissues

[0269] Lung microtissues are cultured with two media, initiation medium and maturation medium, as described in Example 2. Initiation medium is continuously administered until the tissue is deemed to be ready for maturation, typically after 3-5 days of culture for the branching microtissue and 5-7 days for the layer-on-layer microtissue. During the maturation phase, the microtissues are transitioned to an air-liquid interface environment. An air-liquid interface is created by exposing the top of the tissue to air. Maturation medium is only added to the bottom compartment of the well. Culture of the model is to be carried out for at least 21 days before full maturity is achieved. Maturity is verified with the observation of the ciliary beating, or the beating of ciliated cells, and immunostaining. Example 6 - Viral Infection of Layer-on-Layer Models

[0270] PR8-H1N1-mCherry viruses were propagated using Madin-Darby Canine Kidney cells (MDCK) and titrated the virus stock to various dilutions. Virus supernatants exhibiting cytopathic effects and high mCherry expression were collected and stored at -80°C.

[0271] Confirmation of virus infectivity was conducted by infecting MDCK cells. Subsequent confirmation using an agarose overlay showed a titer of approximately 8.5 million plaque forming units per mL (PFU / mL).

[0272] Further validation was performed by infecting layer-on-layer models with the diluted virus. Infection of layer-on-layer models with the diluted virus was carried out by pipetting the virus directly on the model. The models show effective viral infection in both alveolar and bronchial compartments. The layer-on-layer models were infected at day 21 to validate the effectiveness of the model for infection (Figure 13). Viruses were diluted at 1:10, 1:100, and 1:1000 in 200 µL DMEM. These were then added to the permeable inserts and left to incubate for 1.5 hr at 37°C with agitation every 15 min. After viral infection, we removed the viral infection cocktail and let the system in culture under ALI. Viral infection was monitored after 24 and 48 hours post-infection. The presence of mCherry+ cells was observed denoting effective viral infection in both the alveolar and the bronchial compartments. This shows that lung microtissues made according to the present disclosure are useful for the study of viral pathogenesis and host response. Example 7 - WHOLE-MOUNT IMMUNOSTAINING AND IMMUNOFLUORESCENCE OF 3D MACROSCALE TISSUES

[0273] In this study, the protocol for whole-mount immunostaining and immunofluorescence of 3D macroscale lung tissues was meticulously followed to ensure precise and reproducible results. The reagents necessary for this experiment were prepared beforehand, which included the creation of a Perm-Block Buffer by dissolving 5 grams of BSA in 1X PBS and adding 300 µL of Triton X-100 to make a 100 mL solution. Similarly, a Wash Buffer was prepared by mixing 90 mL of PBS, 10 mL of Perm-Block Buffer, and 90 µL of Tween-20. The primary antibody cocktail was diluted in the Perm-Block Buffer and kept on ice, prepared in sufficient volume per sample, typically requiring a resuspension in 1 mL of the cocktail. The secondary antibody and counterstain cocktail were diluted in the Wash Buffer, with secondary antibodies often diluted at ratios between 1:500 to 1:1000, and counterstains like UV nuclear dyes (DAPI or Hoechst 3342) included.

[0274] For microtissue fixation, the culture media from the organoid cultures were aspirated, and the cultures were rinsed twice with 1X PBS. The 3D lung prints were resuspended in 4% PFA, adding 1 mL on both the top and bottom compartments and incubated for 30 minutes at 37°C with agitation. Following this, the PFA was aspirated into appropriate waste containers, and the samples were rinsed with PBS and washed with Wash Buffer for 15 minutes, with this step repeated three times. These fixed samples were stored at 4°C for at least a week before staining, with a suggestion to reconstitute them in a solution containing 0.5% BSA and 2 mM sodium azide in PBS for extended storage to maintain protein epitope stability.

[0275] The next phase involved permeabilization and antibody staining. The buffer was aspirated from the microtissues, and they were resuspended in 1 mL of Perm-Block Buffer, incubated with shaking or rocking for 3 hours at 37°C. Concurrently, the primary antibody cocktail was prepared if it had not been done so already. After 3 hours, the Perm-Block Buffer was aspirated, and 1 mL of the primary antibody cocktail was added on the apical side, covered with parafilm to prevent drying, and incubated at 4°C for 3 to 4 days. For the layer-on-layer model, 48 hours of incubation was sufficient. The antibody cocktail was then aspirated, and the samples were rinsed once using the Wash Buffer. They were washed with 1 mL of Wash Buffer on both top and bottom compartments for 15-30 minutes at room temperature, repeated three times.

[0276] The final steps, performed in the dark, involved adding secondary antibodies and counterstains, followed by a 3-hour incubation at room temperature. The antibody cocktail was aspirated, and the samples were rinsed with the Wash Buffer, followed by a 30-minute incubation at room temperature, repeated three times. A subsequent rinse with 1 mL of PBS allowed for a brief check of the staining effectiveness. For tissue clearing, a mix of 0.5 mL Ce3D buffer with 0.5 mL PBS was added to the top compartment and incubated for 3 hours to overnight at room temperature with shaking. The buffer was then aspirated, and 1 mL of 100% Ce3D buffer was added to a glass-bottom dish, with the microtissue transwell transferred into this dish and an additional 1 mL of Ce3D buffer added on top. This was incubated for at least 3 hours, up to 24 hours, with visual tracking of tissue clearing from opaque to transparent.

[0277] For imaging, a confocal microscope was recommended. Key considerations included consulting the Nyquist sampling dimensions for optimal super-resolution imaging, adjusting pinhole size to lower airy unit values for clearer images, averaging images 3 to 4 times to reduce background noise, and setting laser and detector parameters to minimize pixel oversaturation. For super-resolution imaging, adherence to Nyquist sampling and post- acquisition deconvolution were recommended to resolve sub-micrometer structures like organelles. Example 8 - ORGANOID CULTURE FROM PRIMARY LUNG BRONCHIAL AND ALVEOLAR EPITHELIAL CELLS

[0278] In this study, the protocol for culturing primary lung organoids from bronchial and alveolar epithelial cells was followed. Initially, all necessary materials and reagents were prepared, including the thawing of Cultrex overnight at 4°C and the preparation of basal and expansion media. The Cultrex was adjusted to a concentration of 8 mg / mL, and various stock solutions for the lung organoid expansion media were prepared and stored as required.

[0279] The organoid initiation began with setting the water bath to 37°C and sterilizing the culture hood with 70% ethanol. Cryopreserved cells were removed from liquid nitrogen storage, thawed in the water bath until a small amount of ice remained, and transferred to a conical vial. The cells were then centrifuged, the supernatant discarded, and the cells resuspended in Cultrex. Domes were formed on pre-warmed culture plates and incubated to solidify before being overlaid with lung organoid expansion media.

[0280] Following incubation at 37°C with 5% CO2, organoid growth was monitored, and the cultures were typically ready for differentiation experiments after 10-14 days. For passaging, the density of organoids was assessed microscopically to determine the split ratio. Organoids were harvested, dislodged from their culture plates, and broken down into smaller fragments using TryPLE. After enzymatic digestion, the organoids were resuspended in Cultrex and plated as domes in fresh culture plates, followed by incubation with expansion media.

[0281] For cryopreservation, organoids were examined and harvested while in an immature but healthy state, specifically around 4 days old for alveolar organoids. The organoids were resuspended in media containing 10% DMSO, transferred into cryovials, and initially frozen at -80°C before being moved to long-term storage in liquid nitrogen. Throughout the process, care was taken to maintain cold conditions to ensure high post-thaw viability of the organoids.

[0282] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, and the figures, the foregoing description and figures are 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.

Claims

CLAIMS: What is claimed is:

1. An engineered tissue model, comprising: a permeable membrane having at least one tissue type on a first side, the tissue type comprising a plurality of cells; a support structure on at least a portion of a second side of the permeable membrane; and optionally, a basal layer on the second side of the permeable membrane; wherein: the first side and the second side are opposite each other; the tissue type is derived from stem cells.

2. The engineered tissue model of claim 1, wherein the permeable membrane has a pore size of 0.4 μm – 8.0 μm.

3. The engineered tissue model of claim 1 or claim 2, wherein the permeable membrane has two tissue types on the first side.

4. The engineered tissue model of claim 3, wherein the two tissue types are located in different portions of the permeable membrane.

5. The engineered tissue model of claim 3 or claim 4, wherein the two tissue types are arranged concentrically.

6. The engineered tissue model of any one of claims 1-5, wherein the support structure is plastic or metal.

7. The engineered tissue model of any one of claims 1-6, wherein the basal layer comprises endothelial cells, fibroblast cells, and at least one polymer.

8. The engineered tissue model of claim 7, wherein the endothelial cells are human pulmonary microvascular endothelial cells.

9. The engineered tissue model of claim 7 or claim 8, wherein the polymer is selected from collagen type I, gelatin methacrylate, collagen type I, collagen type IV, solubilized extracellular matrix proteins derived from a basement membrane, solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, and combinations of the foregoing.

10. The engineered tissue model of any one of claims 1-9, wherein the stem cells are alveolar stem cells, bronchial stem cells, or both.

11. A method of making the engineered tissue model of any one of claims 1-10, comprising: coating a first side of a permeable membrane with extracellular matrix proteins; optionally, depositing a sacrificial wall onto the first side of the membrane, the sacrificial wall defining a space within which at least one type of stem cells are cultured; attaching a support structure to at least a portion of a second side of the membrane; optionally, depositing a basal layer on the second side of the membrane; seeding the at least one type of stem cells onto the first side, optionally within the space defined by the sacrificial wall, and culturing the at least one type of stem cells by submerging in an initiation culture medium; and thereafter culturing the at least one type of stem cells in a maturation culture medium via an air-liquid interface culture; wherein: the initiation medium and maturation medium are different; and the air-liquid interface comprises removing the maturation medium from the first side of the membrane while keeping a second side of the membrane in contact with the maturation medium.

12. A method of making a three-dimensional tissue model, comprising: seeding endothelial cells onto a first surface of a permeable membrane and growing the cells to confluence on the first side; contacting the first side with a hydrogel comprising mesenchymal cells, endothelial cells, and at least one polymer; optionally creating a three-dimensional pattern on the first side, within the hydrogel; depositing alveolar cells, bronchial cells, and bronchial smooth muscle cells onto the first side, through the hydrogel, optionally within the three-dimensional pattern; culturing the deposited cells by submerging in an initial culture medium; and thereafter culturing the alveolar cells, bronchial cells, and bronchial smooth muscle cells in a maturation culture medium via an air-liquid interface culture; wherein: the initiation medium and maturation medium are different; andthe air-liquid interface comprises removing the maturation medium from the first side of the membrane while keeping a second side of the membrane in contact with the maturation medium.

13. A three-dimensional tissue model prepared by the method of claim 12.

14. A method of culturing lung epithelial stem cells, comprising: coating at least one surface of a culture vessel with extracellular matrix proteins; contacting the coated surface with undifferentiated lung stem cells in an initiation medium; and culturing the cells in the initiation medium; wherein the initiation medium maintains the undifferentiation for at least 8 passages.

15. The method of any one of claims 11, 12, or 14, wherein the initiation medium comprises: VEGF-165, FGF-2, FGF-7, FGF-10, and EGF; and optionally further comprises: Y27632, CHIR-99021, LDN-193189, A-83-01, SB202190, CHIR-99021, LDN- 193189 hydrochloride, A-83-01, ascorbic acid, and / or SB202190.

16. The method of claim 11 or claim 12, wherein the maturation medium comprises: 10 % serum and agonists for FGFR and VEGFR signaling pathways.

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