Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: Methodology

<p>Abstract</p> <p>Background</p> <p>Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.</p> <p>Results</p> <p>The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior.</p> <p>Conclusions</p> <p>The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.</p

Research on preparation of complex cell systems for new therapies

Im Rahmen dieser Arbeit wurde der Arbeitsablauf zur Präparation komplexer, funktionaler Zellsysteme im Kontext neuer Therapien in die vier Hauptschritte Gewinnung, Aufbereitung, Kryokonservierung und Applikation eingeteilt und hinsichtlich möglicher Schädigungsmechanismen und Optimierungs-möglichkeiten untersucht. Dazu wurde ein Zellpanel aus relevanten Zelltypen mit entsprechenden Modellsystemen entwickelt und ein Algorithmus zur Protokollentwicklung und Methodenwahl abgeleitet. Vergleichende Untersuchungen von Wachstumsoberflächen und neuartigen, miniaturisierten Bioreaktoren mit Automatisierungspotenzial wurden im Hinblick auf Generierung und Verarbeitung funktionaler Mikrogewebe durchgeführt und ihre Einflüsse auf Zellen analysiert. Erstmalig wurden Raum-Zeit-Kurven einschlussimmobilisierter Bioimplantate in Hydrogelkapseln während des Polymerisationsprozesses untersucht und quantifiziert. Unter Berücksichtigung des Erhalts zellspezifischer Funktionalität, wurden neuartige Verfahren zur Kryokonservierung mit Automatisierungspotenzial entwickelt und untersucht. In miniaturisierten, tropfenbasierten Verfahren zur Vitrifikation konnten zellspezifische Eigenschaften sowie eine größtmögliche Flexibilität der Proben für die weitere Applikationen erhalten werden. Zur unmittelbaren Anwendung kryokonservierter Proben wurde eine neuartige Applikationseinheit entwickelt. Ihr Einfluss auf die Funktionalität applizierter Proben wurde anhand therapeutisch relevanter Zellen untersucht.Within this thesis the workflow for the preparation of complex cell systems in new therapies was subdivided into the four main steps retrieval, processing, cryopreservation and application. Each step was examined in terms of damage mechanisms and screened for optimization. Therefore, a cell panel of therapeutically relevant cells and according model systems was established and an algorithm for the selection of protocols and methods was developed. Comparative investigations of growth surfaces and novel miniaturized bioreactors were performed with regard to generation and processing of functional micro tissues. Bioreactor’s potentials for automation was examined, as well as their influences on cells. For the first time, trajectories of immobilized grafts in hydrogel capsules were observed and quantified during the polymerization process. Considering the preservation of functionality, novel cryopreservation methods with the possibility for automation were established and examined. Functionality of cells was preserved in droplet-based vitrification techniques, which kept the maximal flexibility of the frozen sample. A setup was established for the direct application of vitrified samples by air pressure. Its influence on cell’s functionality was investigated by different therapeutically relevant cell systems

Clinical Efficacy of a Spray Containing Hyaluronic Acid and Dexpanthenol after Surgery in the Nasal Cavity (Septoplasty, Simple Ethmoid Sinus Surgery, and Turbinate Surgery)

Background. This prospective, controlled, parallel-group observational study investigated the efficacy of a spray containing hyaluronic acid and dexpanthenol to optimise regular treatment after nasal cavity surgery in 49 patients with chronic rhinosinusitis. Methods. The control group received standard therapy. Mucosal regeneration was determined using rhinoscopy sum score (RSS). Pre- and postoperative nasal patency was tested using anterior rhinomanometry. The participants were questioned about their symptoms. Results. Regarding all RSS parameters (dryness, dried nasal mucus, fibrin deposition, and obstruction), mucosal regeneration achieved good final results in both groups, tending to a better improvement through the spray application, without statistically significant differences during the whole assessment period, the mean values being 7.04, 5.00, 3.66, and 3.00 (intervention group) and 7.09, 5.14, 4.36, and 3.33 (control group). No statistically significant benefit was identified for nasal breathing, foreign body sensation, and average rhinomanometric volume flow, which improved by 12.31% (control group) and 11.24% (nasal spray group). Conclusion. The investigational product may have additional benefit on postoperative mucosal regeneration compared to standard cleaning procedures alone. However, no statistically significant advantage could be observed in this observational study. Double-blind, controlled studies with larger populations will be necessary to evaluate the efficacy of this treatment modality

Application-Oriented Bulk Cryopreservation of Human iPSCs in Cryo Bags Followed by Direct Inoculation in Scalable Suspension Bioreactors for Expansion and Neural Differentiation

Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells' fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.EU(

Zooming in on cryopreservation of hiPSCs and neural derivatives: A dual-center study using adherent vitrification

Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow‐rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post‐thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post‐thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual‐center study, we compared the results by immunocytochemistry (ICC), fluorescence‐activated cell sorting analysis, and RNA‐sequencing (RNA‐seq). Adherent vitrification was achieved in the so‐called TWIST substrate, a device combining cultivation, vitrification, storage, and post‐thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results were not significantly different at day 4 after thawing. RNA‐seq and ICC of hiPSCs revealed no change in gene expression and pluripotency markers, indicating that physical damage of slow‐rate freezing disrupts cellular membranes. Scanning electron microscopy showed preserved colony integrity by adherent vitrification. Experiments using smNPCs demonstrated that adherent vitrification is also applicable to neural derivatives of hiPSCs. Our data suggest that, compared to the state‐of‐the‐art slow‐rate freezing in suspension, adherent vitrification is an improved cryopreservation technique for hiPSCs and derivatives