Mesenchymal stem cell extraction from human umbilical cord tissue: processing to understand and minimise variability in cell yield

Human tissue banks are a potential source of cellular material for the emerging cellbased therapy industry; umbilical cord tissue (UCT) private banking is increasing in such facilities as a source of mesenchymal stem cells for future therapeutic use. However, early handling of UCT is relatively uncontrolled due to the clinical demands of the birth environment and subsequent transport logistics. It is therefore necessary to develop extraction methods that are robust to real world operating conditions,rather than idealised operation. This will be critical for all processes using primary tissue or cell sources. The research work undertaken in this PhD project was initiated by the collaboration with one of the leading private cord blood banks in the UK and later driven by the prospect of expanding the cell therapy and business potential of the bank. The investigation described in this thesis has focused on: - Developing an extraction method for human mesencymal stem cells (hMSCs) from UCT. - Understanding and minimizing the noticed variability in cell yield extracted from UCT by mapping the operating environment and assessing the risk factors before empirically determining their effect on the process. - Establishing the necessary process controls in the production of high quality hMSCs, through a series of wet experiments, targeted at narrowing down the sources of variability down to sub-process level. - Finding a novel method for assessing the cell content and viability of cords prior to processing. Therefore, helping the tissue processing facility to predict the risk of suboptimal cell yield from a given cord tissue section and processing method, given different operating ranges. - Determining the tissue storage requirements and isolation method with acceptable risk of adequate cell recovery. - Characterization of cells extracted from UCT via different extraction methods and comparison to primary cells extracted from other tissue sources. - Investigation of cryopreservation method for UCT. The result of this work provides a solid example of the type of data and analysis that will be required to inform a Quality-by-Design type approach for cell product development and manufacture. It will help tissue processing facilities and banks to predict the probability of cell yields from tissue sections given different operating ranges, and to aid and inform the experimental approach of others

Airway compliance measured by anatomic optical coherence tomography

Quantification of airway compliance can aid in the diagnosis and treatment of obstructive airway disorders by detecting regions vulnerable to collapse. Here we evaluate the ability of a swept-source anatomic optical coherence tomography (SSaOCT) system to quantify airway cross-sectional compliance (CC) by measuring changes in the luminal cross-sectional area (CSA) under physiologically relevant pressures of 10–40 cmH2O. The accuracy and precision of CC measurements are determined using simulations of non-uniform rotation distortion (NURD) endemic to endoscopic scanning, and experiments performed in a simplified tube phantom and ex vivo porcine tracheas. NURD simulations show that CC measurements are typically more accurate than that of the CSAs from which they are derived. Phantom measurements of CSA versus pressure exhibit high linearity (R2>0.99), validating the dynamic range of the SSaOCT system. Tracheas also exhibited high linearity (R2 = 0.98) suggestive of linear elasticity, while CC measurements were obtained with typically ± 12% standard error

Integrated diffuse optical tomography and photoacoustic tomography: phantom validations

We designed, fabricated and tested a novel imaging system that fuses diffuse optical tomography (DOT) and photoacoustic tomography (PAT) in a single platform. This platform takes advantages of both DOT and PAT, and can potentially provide dual-modality two dimensional functional and cellular images of the breast quantitatively. Here we describe this integrated platform along with initial tissue phantom validations

In vivo and in situ cellular imaging full-field optical coherence tomography with a rigid endoscopic probe

Full-field OCT has proved to be a powerful high-resolution cellular imaging tool for biological tissues. However the standard bulk full-field OCT setup does not match the size requirements for most in situ and in vivo imaging applications. We adapted its principle into a rigid needle-like probe using two coupled interferometers and incoherent illumination: an external processing interferometer is used for in-depth scanning, while a distal common-path interferometer at the tip of the probe collects light backscattered from the tissue. Our experimental setup achieves an axial and transversal resolution in tissue of 1.8 µm and 3.5 µm respectively, for a sensitivity of −80 dB. We present ex vivo images of human breast tissue, and in vivo images of different areas of human skin, which reveal cellular-level structures

A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies

Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow–derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables

Automated algorithm for breast tissue differentiation in optical coherence tomography

Abstract. An automated algorithm for differentiating breast tissue types based on optical coherence tomography ͑OCT͒ data is presented. Eight parameters are derived from the OCT reflectivity profiles and their means and covariance matrices are calculated for each tissue type from a training set ͑48 samples͒ selected based on histological examination. A quadratic discrimination score is then used to assess the samples from a validation set. The algorithm results for a set of 89 breast tissue samples were correlated with the histological findings, yielding specificity and sensitivity of 0.88. If further perfected to work in real time and yield even higher sensitivity and specificity, this algorithm would be a valuable tool for biopsy guidance and could significantly increase procedure reliability by reducing both the number of nondiagnostic aspirates and the number of false negatives

Combined anatomical optical coherence tomography and intraluminal pressure reveal viscoelasticity of the in vivo airway

It is hypothesized that the local, viscoelastic (time-dependent) properties of the airway are important to accurately model and ultimately predict dynamic airway collapse in airway obstruction. Toward this end, we present a portable, endoscopic, swept-source anatomical optical coherence tomography (aOCT) system combined with a pressure catheter to capture local airway dynamics in vivo during respiration. aOCT scans were performed in the airways of a mechanically ventilated pig under paralysis with dynamic and static ventilation protocols. Validation of dynamic aOCT luminal cross-sectional area (CSA) measurements against Cine CT, obtained during the same exam, showed an aggregate difference of 15 % ± 3 %. aOCT-derived CSA obtained in the in vivo trachea also exhibited hysteresis as a function of pressure, depicting the viscoelastic nature of the airway wall. The volumetric imaging capabilities were validated by comparing aOCT- and CT-derived geometries of the porcine airway spanning nine generations from the trachea to the bronchioles. The ability to delineate regional differences in airway viscoelastic properties, by measuring airway deformation using aOCT combined with intraluminal pressure, paves the way to patient-specific models of dynamic airway collapse

Three dimensional tracking for volumetric spectral-domain optical coherence tomography

We present a three-dimensional (3D) tracker for a clinical ophthalmic spectral domain optical coherence tomography (SD-OCT) system that combines depth-tracking with lateral tracking, providing a stabilized reference frame for 3D data recording and post acquisition analysis. The depth-tracking system is implemented through a real-time dynamic feedback mechanism to compensate for motion artifact in the axial direction. Active monitoring of the retina and adapting the reference arm of the interferometer allowed the whole thickness of the retina to be stabilized to within +/- 100 mu m. We achieve a relatively constant SNR from image to image by stabilizing the image of the retina with respect to the depth dependent sensitivity of SD-OCT. The depth tracking range of our system is 5.2 mm in air and the depth is adjusted every frame. Enhancement in the stability of the images with the depth-tracking algorithm is demonstrated on a healthy volunteer.X1119sciescopu

Label-free imaging of zebrafish larvae in vivo by photoacoustic microscopy

Zebrafish play an important role in biological and biomedical research. Traditional in vivo imaging methods for studying zebrafish larvae primarily require fluorescence labeling. In this work, relying on tissue intrinsic optical absorption contrast, we acquired high resolution label-free 3D images of zebrafish larvae by using photoacoustic microscopy (PAM) in vivo. The spatial resolution reaches several microns, allowing the study of microstructures in various living organs. We demonstrated that our method has the potential to be a powerful non-invasive imaging method for studying various small animal models, including zebrafish larvae, Caenorhabditis elegans, frogs and drosophila larvae