Tissue Engineering, Part C: Methods
ISSN / EISSN: 19373384 / 19373392
Published by: Mary Ann Liebert Inc
Total articles ≅ 1,395
Latest articles in this journal
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0184
The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections, and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome (ARDS), pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, though are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a 3D in vitro lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions, and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such an LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until seven days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues, and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0189
The advent of single-cell RNA sequencing (scRNA-Seq) has brought with it the ability to gain greater insights into the cellular composition of tissues and heterogeneity in gene expression within specific cell types. For tissue engineered blood vessels, this is particularly impactful to better understand how neotissue forms and remodels into tissue resembling a native vessel. A notable challenge, however, is the ability to separate cells from synthetic biomaterials to generate high quality single-cell suspensions to interrogate the cellular composition of our tissue engineered vascular grafts during active remodeling in situ. We present here a simple, commercially available approach to separate cells within our tissue engineered vascular graft (TEVG) from the residual scaffold for downstream use in a scRNA-Seq workflow. Utilizing this method, we identified the cell populations comprising explanted TEVGs and compared these with results from immunohistochemical analysis. The process began with explanted TEVGs undergoing traditional mechanical and enzymatic dissociation to separate cells from scaffold and extracellular matrix proteins. Magnetically labelled antibodies targeting murine origin cells were incubated with enzymatic digests of TEVGs containing cells and scaffold debris in suspension allowing for separation by utilizing a magnetic separator column. Single-cell suspensions were processed through 10x Genomics and data were analyzed utilizing R to generate cell clusters. Expression data provided new insights into a diverse composition of phenotypically unique subclusters within the fibroblast, macrophage, smooth muscle cell, and endothelial cell populations contributing to the early neotissue remodeling stages of TEVGs. These populations were correlated qualitatively and quantitatively with immunohistochemistry highlighting for the first time the potential of scRNA-Seq to provide exquisite detail into the host cellular response to an implanted TEVG. These results additionally demonstrate magnetic cell isolation is an effective method for generating high quality cell suspensions for scRNA-Seq. While this method was utilized for our group’s tissue engineered vascular grafts, it has broader applications to other implantable materials that use biodegradable synthetic materials as part of scaffold composition.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0181
Engineering clinically relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large-scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies which support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this paper, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0200
Chronic Kidney Disease (CKD) is the irreversible loss of nephron function, leading to a build-up of toxins, prolonged inflammation, and ultimately fibrosis. Currently, no effective therapies exist to treat CKD due to its complex pathophysiology. Mesenchymal stem/stromal cell (MSC) transplantation is a promising strategy to treat kidney diseases, and multiple clinical trials are currently ongoing. We previously demonstrated that rat bone marrow-derived MSC (BMSC) sheets transplanted onto surgically de-capsulated kidney exert therapeutic effects that suppressed renal fibrosis progression based on enhanced vascularization. However, there are clinical concerns about kidney decapsulation such as impaired glomerular filtration rate and Na+ ion and H2O excretion, leading to kidney dysfunction. Therefore, for transitioning from basic research to translational research using cell sheet therapy for kidney disease, it is essential to develop a new cell sheet transplantation strategy without kidney decapsulation. Significantly, we employed cell sheets engineered from clinical-grade human clonal BMSC (cBMSC) and transplanted these onto intact renal capsule to evaluate their therapeutic ability in the rat ischemia reperfusion injury (IRI) model. Histological analysis 1-day post-surgery showed that cBMSC sheets engrafted well onto intact renal capsule. Interestingly, some grafted cBMSCs migrated into the renal parenchyma. At 1- to 3-days post-surgery (acute stage), grafted cBMSC sheets prevented tubular epithelial cell injury. At 28-days post-surgery (chronic phase), we observed that grafted cBMSC sheets suppressed renal fibrosis in the rat IRI model. Taken together, engineered cBMSC sheet transplantation onto intact renal capsule suppresses tubular epithelial cell injury and renal fibrosis, supporting further development as a possible clinically relevant strategy.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0207
Local tumor treatment is a feasible measure for patients with glioblastoma (GBM) who are unsuitable for surgical resection. Interferon-elastin-like polypeptide (IFN-ELP(V)) is a slow-release, biodegradable, thermosensitive fusion protein with antitumor immunity, and resveratrol (Res) is a polyphenolic compound with an antitumor effect. In this study, we found that intratumor injection of IFN-ELP(V) combined with intraperitoneal injection of Res is more effective in delaying GBM growth in mice. Specifically, in an orthotopic GBM model, we found a significant improvement in the median survival with this strategy. Our results suggested that the combined use of IFN-ELP(V) and Res has a dramatic synergistic effect on GBM, thus providing a novel and effective therapeutic strategy for tumors.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0214
Three-dimensional (3D) bioprinting demonstrates technology capable of producing structures comparable to native tissues in the human body. The Freeform Reversible Embedding of Suspended Hydrogels (FRESH) technique involves hydrogel-based bio-inks printed within a thermo-reversible support bath to provide mechanical strength to the printed construct. Smaller and more uniform microsphere sizes of FRESH were reported to aid in enhancing printing resolution and construct accuracy. Therefore, we sought to optimize the FRESH generation protocol, particularly by varying stir speed and stir duration, in hopes to further improve microsphere size and uniformity. We observed optimal conditions at a stir speed of 600 rpm and stir duration for 20 hours that generated the smallest microspheres with the best uniformity. Comparison of using the optimized FRESH to the commercial FRESH LifeSupport to bioprint single filament and geometrical constructs revealed reduced single filament diameters and higher angular precision in the optimized FRESH bio-printed constructs compared to those printed in the commercial FRESH. Overall, our refinement of the FRESH manufacturing protocol represents an important step toward enhancing 3D bioprinting resolution and construct fidelity. Improving such technologies allows for the fabrication of highly accurate constructs with anatomical properties similar to native counterparts. Such work has significant implications in the field of tissue engineering for producing accurate human organ model systems.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0193
Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase-levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pre-treated and un-treated donor. Furthermore, the transcription levels of CYP 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens prior to isolation seem to perform as well as hepatocytes without pre-operative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy prior to liver resection are an uncompromised resource for pharmacologic and toxicologic studies.
Tissue Engineering, Part C: Methods; https://doi.org/10.1089/ten.tec.2022.0159
Mesenchymal stem cells (MSC) stimulate nerve and tissue regeneration and are primed for clinical translation. Application of autologous MSCs is limited by requirements for expedient harvesting procedures, proliferative expansion to increase cell numbers, and reduced regenerative potential due to aging or pathological conditions. Because MSCs are immune-privileged, allogeneic MSCs may serve as “off-the-shelf” cell-based reconstructive treatments to support nerve repair. Therefore, we examined the safety and immune response parameters of allogeneic MSCs seeded on NeuraGen® Nerve Guides (NNG) in a rabbit model. NNGs with or without allogeneic rabbit MSCs were applied to rabbit sciatic nerves. Randomly assigned treatment included: group I (no surgery control, n = 3) or groups II and III (sciatic nerve wrapped with unseeded or allogeneic MSC-seeded NNGs; n = 5/group). Rabbits were euthanized after 2 weeks to monitor functional recovery by histological evaluation of sciatic nerves and tibialis anterior (TA) muscle. Host reactions to allogeneic MSCs were analysed by assessment of body and tissue weight, temperature, as well as haematological parameters, including white blood cell count (WBC), spleen histology, and CD4+ and CD8+ T lymphocytes. Histological analyses of nerves and spleen were all unremarkable, consistent with absence of overt systemic and local immune responses upon allogeneic MSC administration. No significant differences were observed in WBC or CD4+ and CD8+ T lymphocytes across unseeded and seeded treatment groups. Thus, allogenic MSCs are safe for use and may be considered in lieu of autologous MSCs in translational animal studies as the basis for future clinical nerve repair strategies. Impact statement Autologous mesenchymal stem cells (MSC) have been reported to enhance nerve regeneration when used in conjunction with nerve graft substitutes. However, autologous stem cell sources delay treatment and may be susceptible to age- or disease-related dysfunctions. In this work, we investigated the safety of allogeneic MSCs and the optimal cell number for nerve conduit delivery in a rabbit model. When compared to unseeded nerve conduits, allogeneic MSC-seeded conduits did not induce a systemic or local immune response. The findings of this study will ultimately facilitate the
Tissue Engineering, Part C: Methods, Volume 29, pp 30-40; https://doi.org/10.1089/ten.tec.2022.0176
In native articular cartilage, chondrocytes are completely capsulated by a pericellular matrix (PCM), together called the chondron. Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and chondron may be important in regenerative strategies. The current gold standard for the isolation of chondrons from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for chondrocytes and chondrons and characterize the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-hour gold standard chondron isolation method (0.3% dispase + 0.2% collagenase II), an overnight chondron isolation (0.15% dispase + 0.1% collagenase II), and an overnight chondrocyte isolation (0.15% collagenase II + 0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of chondrocytes and chondrons for all three methods. Although the 5-hour chondron isolation resulted in the highest percentage of chondrons, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize chondron yield and homogeneity, the overnight chondron enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting.
Tissue Engineering, Part C: Methods, Volume 29, pp 11-19; https://doi.org/10.1089/ten.tec.2022.0161
Acquired anterior glottic webs (AGW) can lead to abnormally elevated phonatory pitch, dysphonia, and airway obstruction requiring urgent intervention. Herein, we construct a novel AGW rabbit model using heat injury by a laryngoscopic way. A primary study was conducted to identify the injury depth in rabbits’ vocal folds (VFs) by graded heat energy and the heat energy for the incurrence of epithelial layer, lamina propria and muscular layer injury was 25, 30 and 35 W, respectively. Then four different models were designed based on the depth and degree of the injury to determine the optimal procedure for AGW formation. Morphological features, vibratory capacity and histopathological features of the AGW were respectively evaluated. The procedure for conferring the heat injury to the depth of muscular layer and extent of anterior commissure and middle part of bilateral VFs showed the highest success rate of AGW formation (95%, 19/20). For its low cost, effectiveness, and stability for AGW formation, the heat-injury rabbit model with a laryngoscopic approach may provide a new platform for testing novel anti-adhesion materials and bioengineered therapies.