production-human-neurogenin-2-inducible-neurons-three-dimensional

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...

Fraunhofer Institute for Biomedical Engineering IBMT

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.

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

We present a novel optical device developed for the monitoring of dynamic behavior in extended 3D-tissue models in various culture environments based on variations in their speckle patterns. The results presented point out the benefit of the technology in terms of detection, accuracy, sensitivity and a reasonable read-out speed as well as reproducibility for the measurements and monitoring of cardiac contractions. We show that the optical read-out technology is suitable for long time monitoring and for drug screening. The method is discussed and compared to other techniques, in particular calcium imaging. The device is flexible and easily adaptable to 2D and 3D-tissue model screenings using different culture environments. The technology can be parallelized for automated read-out of different multi-well-plate formats.

Diffraction-based technology for the monitoring of contraction dynamics in 3D and 2D tissue models.

The European Bank for induced Pluripotent Stem Cells (EBiSC), a non-profit repository for storage, banking, Quality Control (QC) and subsequent distribution of research-grade human induced Pluripotent Stem Cell (iPSC) lines, has centralised iPSC lines generated internationally across >35 disease areas and made them available to users via the EBiSC Catalogue, for research use (cells.ebisc.org/). Comprehensive datasets are accessible prior to purchase detailing the disease background of the original tissue sample, background of iPSC reprogramming and cell line characterisation data. EBiSC also performs robust QC screening to ensure supply of reliable, well-characterised iPSC lines, compliant with ISO9001:2015 principles. Whole Genome Sequencing data for specific iPSC lines can be downloaded from the European Genome Archive, subject to application to the EBiSC Data Access Committee. The EBiSC Access and Use Agreement, required to be completed prior to shipping, can be downloaded from the website along with specific Cell Line Information Packs; together these documents clarify how EBiSC lines can be used for research and detail any specific Third Party Obligations and/or restrictions for use which may apply. A protocol for how to culture and monitor iPSC lines including implementation of routine cell line screening is also available. A second project phase will continue collecting iPSC lines generated internationally, provide iPSC derived differentiated products using improved automation strategies for upscaling and develop the current services provided by EBiSC, including iPSC reprogramming, gene-editing and characterisation.

The EBiSC iPSC bank for disease studies.

The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.

Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity.

To provide a standardized protocol for large-scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC).

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system.

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools.

Scalable expansion of iPSC and their derivatives across multiple lineages.

Hepatocytes are of special interest in biomedical research for disease modelling, drug screening and in vitro toxicology. Human induced pluripotent stem cell (hiPSC)-derived hepatocytes could complement primary human hepatocytes due to their capability for large-scale expansion. In this study, we present an optimized protocol for the generation of hepatocyte-like cells (HLCs) from hiPSC in monolayer (2D) and suspension culture (3D) for production of organoids. A protocol was initially optimized in 2D using a gene edited CYP3A4 Nanoluciferase reporter hiPSC line for monitoring the maturity of HLCs and cryopreservation of definitive endoderm (DE) cells. The protocol was optimized for microwell cultures for high-throughput screening to allow for a sensitive and fast readout of drug toxicity. To meet the increasing demand of hepatic cells in biomedical research, the differentiation process was furthermore translated to scalable suspension-based bioreactors for establishment of hepatic organoids. In pilot studies, the technical settings have been optimized by adjusting the initial seeding density, rotation speed, inoculation time, and medium viscosity to produce homogeneous hepatic organoids and to maximize the biomass yield (230 organoids/ml). To speed up the production process, cryopreservation approaches for the controlled freezing of organoids were analysed with respect to cell recovery and marker expression. The results showed that cryopreserved organoids maintained their phenotype only when derived from hepatocyte progenitors (HPs) at day 8 but not from more mature stages. The establishment of robust protocols for the production of large batches of hepatocytes and hepatic organoids could substantially boost their use in biomedical and toxicology studies.

Article	Year
Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation. PloS one\| PubMed ID: 30682146	2019
Diffraction-based technology for the monitoring of contraction dynamics in 3D and 2D tissue models. Biomedical optics express\| PubMed ID: 32206385	2020
The EBiSC iPSC bank for disease studies. Stem cell research\| PubMed ID: 33099110	2020
Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity. Cryobiology\| PubMed ID: 34582849	2021
Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system. Cell proliferation\| PubMed ID: 35102634	2022
Scalable expansion of iPSC and their derivatives across multiple lineages. Reproductive toxicology (Elmsford, N.Y.)\| PubMed ID: 35595152	2022
Human iPSC-derived hepatocytes in 2D and 3D suspension culture for cryopreservation and in vitro toxicity studies. Reproductive toxicology (Elmsford, N.Y.)\| PubMed ID: 35598806	2022

Julia C. Neubauer

Biography

JoVE Journal Publications

Other Publications