Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

Cardiovascular diseases (CVD) are the leading cause of death around the world, being responsible for 31.8% of all deaths in 2017. 1 The leading cause of CVD is Ischemic heart disease (IHD), which caused 8.1 million deaths in 2013. 2 IHD occurs when coronary arteries in the heart are narrowed or blocked, preventing the flow of oxygen and blood into the cardiac muscle, which could provoke acute myocardial infarction (AMI) and ultimately lead to heart failure and death. Cardiac regenerative therapy aims to repair and refunctionalize damaged heart tissue through the application of (1) intramyocardial cell delivery, (2) epicardial cardiac patch, and (3) acellular biomaterials. These approaches have provided benefit of cell localization and tissue structure respectively. However, to improve cell retention and integration, there is a need for the intramyocardial delivery of functional tissues while preserving anisotropic muscle alignment. Here, we developed a biodegradable z-wire scaffold that supports the scalable gel-free production of an array of functional cardiac tissues in a 384-well plate format. The z-wire scaffold design supports cellular alignment, provides tunable mechanical support, and allows for hallmark tissue contraction. When the scaffold is imparted with magnetic properties, individual tissues can be assembled with macroscopic alignment under magnetic guidance. When used in combination with a customized surgical delivery tool, z-wire tissues can be injected directly into the myocardial wall, with controlled tissue orientation according to the injection path. This modular tissue engineering approach, in combination with the use of smart scaffolds, could expand opportunity in functional tissue delivery.

Background:Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC).Purpose:To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC.Study Design:Descriptive laboratory study.Methods:Two-dimensional cultures of human AC–derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm–thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.Results:Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC.Conclusion:This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage tissue with properties similar to the deep layer of HC in vitro.Clinical Relevance:The HC tissue obtained by the method described can be used to develop an implantable product for the replacement of damaged or malformed AC, especially in younger patients where the lesions are caused by trauma or mechanical stress.

The histocytochemical and molecular analysis of cells that constitute the aortic valve (AV) of the rat heart was done in this study. We have focussed on the identity of cells in the spongiosal layer of the valve by immunofluorescence studies using lineage specific markers and cytochemical staining. We have established two-dimensional (2D) cultures of cells from isolated rat AV leaflets and maintained endothelial and interstitial valvular cells (IVC) over a period of six to eight weeks. Using "passage 0" cells from 2D valvular cultures, we could reconstruct the three-dimensional (3D) valvular tissue in collagen gels that showed very similar cellular organization and marker expression profile, as that of the native tissue. Lineage specific markers in the native tissue and cell cultures were studied by Reverse Transcriptase-PCR and immunofluorescence for VCAM-I, -SMA, collagen I, CD71, collagen II, and Ecadherin markers. This is the first report on the identification of cell lineages in the spongiosal layer of AV and the successful reconstruction of 3D valvular tissue from primary cell cultures of AV.

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