Rapid and Continuous Cryopreservation of Stem Cells with a 3D Micromixer.

Cryopreservation is the final step of stem cell production before the cryostorage of the product. Conventional methods of adding cryoprotecting agents (CPA) into the cells can be manual or automated with robotic arms. However, challenging issues with these methods at industrial-scale production are the insufficient mixing of cells and CPA, leading to damage of cells, discontinuous feeding, the batch-to-batch difference in products, and, occasionally, cross-contamination. Therefore, the current study proposes an alternative way to overcome the abovementioned challenges; a highly efficient micromixer for low-cost, continuous, labour-free, and automated mixing of stem cells with CPA solutions. Our results show that our micromixer provides a more homogenous mixing of cells and CPA compared to the manual mixing method, while the cell properties, including surface markers, differentiation potential, proliferation, morphology, and therapeutic potential, are well preserved.

A modular 3D printed microfluidic system: a potential solution for continuous cell harvesting in large-scale bioprocessing.

Microfluidic devices have shown promising applications in the bioprocessing industry. However, the lack of modularity and high cost of testing and error limit their implementation in the industry. Advances in 3D printing technologies have facilitated the conversion of microfluidic devices from research output to applicable industrial systems. Here, for the first time, we presented a 3D printed modular microfluidic system consisting of two micromixers, one spiral microfluidic separator, and one microfluidic concentrator. We showed that this system can detach and separate mesenchymal stem cells (MSCs) from microcarriers (MCs) in a short time while maintaining the cell's viability and functionality. The system can be multiplexed and scaled up to process large volumes of the industry. Importantly, this system is a closed system with no human intervention and is promising for current good manufacturing practices.

Bronchial brushings for investigating airway inflammation and remodelling.

Asthma is the commonest medical cause for hospital admission for children in Australia, affects more than 300 million people worldwide, and is incurable, severe in large number and refractory to treatment in many. However, there have been no new significant treatments despite intense research and billions of dollars. The advancement in our understanding in this disease has been limited due to its heterogeneity, genetic complexity and has severely been hampered particularly in children by the difficulty in obtaining relevant target organ tissue. This review attempts to provide an overview of the currently used and recently developed/adapted techniques used to obtain lung tissue with specific reference to the airway epithelium.

Mussel inspired ZIF8 microcarriers: a new approach for large-scale production of stem cells.

Metal-organic frameworks (MOFs) have high porosity, large surface area, and tunable functionality and have been widely used for drug loading. The aim of this study was to exploit unique features of zeolitic imidazolate framework-8 (ZIF8) to develop an innovative composite microcarrier (MC) for human mesenchymal stem cells (hMSCs) adhesion and proliferation. ZIF8 MCs were prepared by immobilization of polydopamine/polyethyleneimine (PDA/PEI) and ZIF8 on the surface of polystyrene beads. The chemical properties of MCs such as coating stability and homogeneity were characterized by different techniques such as ATR-FTIR, XRD, EDX, SEM, and contact angle. The prepared MCs were tested using human adipose-derived mesenchymal stem cells (hADSCs) in both static and dynamic conditions, and results were compared to a commercially available MC (Star-Plus), polydopamine coated MCs and amine-functionalized MCs as a control. Results show that PDA/PEI/ZIF8 coated MCs (in short: ZIF8 MCs) provides an excellent biocompatible environment for hADSCs adhesion and growth. In conclusion, ZIF8 MCs represent suitable and low-cost support for hADSCs culture and expansion, which can maximize cell yield and viability while preserving hADSCs multipotency. The present findings have revealed this strategy has the potential for chemical and topographical modification of MCs in tissue engineering applications.

Azithromycin Partially Mitigates Dysregulated Repair of Lung Allograft Small Airway Epithelium.

BACKGROUND: Dysregulated airway epithelial repair following injury is a proposed mechanism driving posttransplant bronchiolitis obliterans (BO), and its clinical correlate bronchiolitis obliterans syndrome (BOS). This study compared gene and cellular characteristics of injury and repair in large (LAEC) and small (SAEC) airway epithelial cells of transplant patients. METHODS: Subjects were recruited at the time of routine bronchoscopy posttransplantation and included patients with and without BOS. Airway epithelial cells were obtained from bronchial and bronchiolar brushing performed under radiological guidance from these patients. In addition, bronchial brushings were also obtained from healthy control subjects comprising of adolescents admitted for elective surgery for nonrespiratory-related conditions. Primary cultures were established, monolayers wounded, and repair assessed (±) azithromycin (1 µg/mL). In addition, proliferative capacity as well as markers of injury and dysregulated repair were also assessed. RESULTS: SAEC had a significantly dysregulated repair process postinjury, despite having a higher proliferative capacity than large airway epithelial cells. Addition of azithromycin significantly induced repair in these cells; however, full restitution was not achieved. Expression of several genes associated with epithelial barrier repair (matrix metalloproteinase 7, matrix metalloproteinase 3, the integrins ß6 and ß8, and ß-catenin) were significantly different in epithelial cells obtained from patients with BOS compared to transplant patients without BOS and controls, suggesting an intrinsic defect. CONCLUSIONS: Chronic airway injury and dysregulated repair programs are evident in airway epithelium obtained from patients with BOS, particularly with SAEC. We also show that azithromycin partially mitigates this pathology.

Successful establishment of primary small airway cell cultures in human lung transplantation.

BACKGROUND: The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation. METHODS: SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP). RESULTS: The cell yield for SAEC (0.956 +/- 0.063 x 106) was lower than for large airway cells (1.306 +/- 0.077 x 106) but did not significantly impact on the culture establishment rate (79.0 +/- 5.2% vs. 83.8 +/- 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded. CONCLUSION: Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD.

Regional differences in susceptibiity of bronchial epithelium to mesenchymal transition and inhibition by the macrolide antibiotic azithromycin.

OBJECTIVE: Dysregulated repair following epithelial injury is a key forerunner of disease in many organs, and the acquisition of a mesenchymal phenotype by the injured epithelial cells (epithelial to mesenchymal transition, EMT) may serve as a source of fibrosis. The macrolide antibiotic azithromycin and the DNA synthesis inhibitor mycophenolate are in clinical use but their mechanism of action remains unknown in post-transplant bronchiolitis obliterans syndrome (BOS). Here we determined if regional variation in the EMT response to TGFß1 underlies the bronchiolocentric fibrosis leading to BOS and whether EMT could be inhibited by azithromycin or mycophenolate. METHODS/RESULTS: We found that small and large airway epithelial cells from stable lung transplant patients underwent EMT when stimulated with TGFß1, however mesenchymal protein expression was higher and loss of epithelial protein expression more complete in small airway epithelial cells. This regional difference was not mediated by changes in expression of the TGFßRII or Smad3 activation. Azithromycin potentially inhibited EMT in both small and large airway epithelial cells by inhibiting Smad3 expression, but not activation. CONCLUSION: Collectively, these observations provide a biologic basis for a previously unexplained but widely observed clinical phenomena, and a platform for the development of new approaches to fibrotic diseases.

The airway epithelium is a direct source of matrix degrading enzymes in bronchiolitis obliterans syndrome.

BACKGROUND: Long-term survival after lung transplantation is hindered by the development of bronchiolitis obliterans syndrome (BOS), and recent evidence suggests that dysregulated epithelial repair may underlie its development. Because matrix metalloproteinase (MMP) -2 and MMP-9 secretion is integral to repair, we hypothesized that airway epithelial cells from patients with BOS would over-express these matrix-degrading enzymes. METHODS: Cells obtained from bronchial and bronchiolar brushings from patients with and without BOS (without acute rejection or infection) were analyzed via quantitative polymerase chain reaction and immunocytochemistry for MMP-2, and MMP-9 gene and protein expression. The expression of tissue inhibitor of metalloproteinase (TIMP)2 and TIMP1 was also assessed. MMP activity in bronchoalveolar lavage was determined via gelatin zymography. RESULTS: MMP-2 and MMP-9 production was significantly higher in bronchoalveolar lavage (3.85- and 11.59-fold, p < 0.001) and airway epithelium (MMP-2 bronchial: 6.33-fold, bronchiolar: 3.57-fold, both p < 0.001; MMP-9 bronchial: 32.55-fold, p < 0.001; bronchiolar: 8.60-fold, p = 0.01) in patients with BOS, but expression in patients without BOS was not different from healthy controls. TIMP expression was similar in patients with and without BOS. Immunostaining confirmed that the airway epithelium was a direct source of MMP-2 and MMP-9 expression in patients with BOS. CONCLUSION: In patients with BOS, the airway epithelium over-expresses MMPs, even in the absence of acute rejection or infection. Dysregulated epithelial repair may be a key feature of BOS.

Cells of epithelial lineage are present in blood, engraft the bronchial epithelium, and are increased in human lung transplantation.

BACKGROUND: There is a growing expectation that cell-based therapies will prove effective for a wide range of conditions including lung diseases such as cystic fibrosis. The promise of these therapies will depend largely on effective delivery and engraftment. In this study, in the setting of human lung transplantation, we sought to determine whether exogenous epithelial cells are able to engraft the transplanted organ and if cells of a similar phenotype could be detected in peripheral blood. METHODS: Cells obtained from bronchial brushings and peripheral blood were analyzed via dual fluorescent in situ hybridization/fluorescent immunohistochemistry (FISH/IHC), short tandem repeat polymerase chain reaction (STR-PCR) and flow cytometry. RESULTS: In 2 of 3 gender-mismatched patients we observed limited (5.9% to 6.8% by STR-PCR and 3.5% to 4% by FISH/IHC) engraftment of the bronchial epithelium by exogenous epithelial cells. Engrafting cells were CD34(-) CD15(-) CD68(-) c-Kit(-), but expressed CXCR4 on the cell surface. Cells with a similar phenotype were also identified in peripheral blood. In 8 patients, at 2 to 66 months post-transplant, 0.57 +/- 0.17% of CD14(-) peripheral blood mononuclear cells were of epithelial lineage. Almost all were CD45(+) and most expressed CXCR4 on the cell membrane. Cells of epithelial lineage were also identified in peripheral blood in healthy individuals but in much lower numbers (0.08 +/- 0.01%, p < 0.05). CONCLUSIONS: Cells of epithelial lineage are detectable in peripheral blood and are able to engraft the bronchial epithelium in humans. Cell numbers are increased in lung transplantation.