Comparability & reimbursement for the translation of scalable, automated stem cell cultures

The research in this thesis focuses primarily on two critical challenges that inhibit the late stage translation of cell-based therapies and Regenerative Medicines (RMs). These include product comparability after a change in manufacturing process or site; and the reimbursement of RMs, in particular those which target multiple simultaneous indications, or Multimorbidity . The automation and standardisation of stem cell cultures also represent key themes of this thesis, which may facilitate the development of scalable, reproducible manufacturing processes for cell-based therapies. Furthermore, given the current uncertainty regarding the characterisation and potency of Human Mesenchymal Stromal (or Stem) Cells (hMSCs) that has inhibited the successful clinical translation of hMSC-based products, understanding the characterisation and putative modes of action of these cells was also a priority throughout this research. Also, due to the increasing number of Human Embryonic Stem Cell (hESC) derived therapies progressing towards market, and the industry-wide shift towards Human Induced Pluripotent Stem Cells (hiPSC) as an alternative to hESCs, the measurement of the growth and characterisation of these cells types represents an important method of demonstrating product comparability after alternative manufacturing process steps in the present thesis. Finally, due to the potential of hiPSCs as a source of large numbers of hMSCs, the culture conditions required to direct the differentiation of hiPSCs to hMSCs are explored

Porous silicon nanoneedles modulate endocytosis to deliver biological payloads

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies

Electric pulses: a flexible tool to manipulate cytosolic calcium concentrations and generate spontaneous-like calcium oscillations in mesenchymal stem cells

Human adipose mesenchymal stem cells (haMSCs) are multipotent adult stem cells of great interest in regenerative medicine or oncology. They present spontaneous calcium oscillations related to cell cycle progression or differentiation but the correlation between these events is still unclear. Indeed, it is difficult to mimic haMSCs spontaneous calcium oscillations with chemical means. Pulsed electric fields (PEFs) can permeabilise plasma and/or organelles membranes depending on the applied pulses and therefore generate cytosolic calcium peaks by recruiting calcium from the external medium or from internal stores. We show that it is possible to mimic haMSCs spontaneous calcium oscillations (same amplitude, duration and shape) using 100 μs PEFs or 10 ns PEFs. We propose a model that explains the experimental situations reported. PEFs can therefore be a flexible tool to manipulate cytosolic calcium concentrations. This tool, that can be switched on and off instantaneously, contrary to chemicals agents, can be very useful to investigate the role of calcium oscillations in cell physiology and/or to manipulate cell fate

Spheroid development for in vitro Chondrogenesis: preliminary studies

This thesis presents the first set of experimental work done at CQM – Centro de Química da Madeira aimed at the implementation of a new research line focused on spheroids for chondrogenesis study. Three different techniques for spheroid preparation using human mesenchymal stem cells (hMSCs) were tested. The preparation of spheroids by cell self-aggregation in ultra-low adhesion (ULA) cell culture plates was shown to be easy to use, and to lead to stable spheroids. The addition of 50 ng/mL carboxymethyl cellulose (CMC) to the cell culture medium helped to control spheroid’s size and spheroidicity, improving cell viability as well. The influence of different culture medium compositions on cell metabolic activity and on cell differentiation towards the chondrogenic lineage was evaluated in 2D cell cultures. The addition of TGF-β1, as well as other supplements, to the chondrogenic medium (CM) increased cell proliferation, and induced chondrogenesis (revealed through histochemical staining). For the assessment of the chondrogenesis in 3D constructs, spheroids were initially prepared and matured for 14 days (50 ng/mL of CMC was added to the medium at day 5) in ULA plates (poly-HEMA coating) in BM with high glucose content, at a high cell seeding density. Then, a spheroid group was exposed to CM; and another maintained in Basal medium (BM), as a control (CTRL) group for more 28 days (chondrogenesis induction). Spheroid’s growth was observed in the CM group, whereas their shrinking was evident in CTRL group. Also, CM led to higher cell viability values in spheroids. Histochemistry staining experiments were only successful using the Fast Green/Safranin O system, revealing the occurrence of chondrogenesis in spheroids exposed to CM. ALP activity measurements showed the absence of the hypertrophic phenotype in the differentiated spheroids (chondrospheres). Finally, ELISA assays allowed the detection of collagen type II and aggrecan in the chondrospheres cultured in CM.Esta tese apresenta o primeiro conjunto de trabalhos experimentais realizados no CQM – Centro de Química da Madeira com vista à implementação de uma nova linha de investigação centrada nos esferóides para o estudo da condrogénese. Foram testadas três técnicas diferentes para a preparação de esferóides utilizando células estaminais mesenquimais humanas (hMSCs). A preparação de esferóides por auto agregação celular em placas de cultura celular de aderência ultra-baixa (ULA) demonstrou ser acessível, conduzindo a esferóides estáveis. A adição de 50 ng/mL de carboximetilcelulose (CMC) ao meio de cultura celular ajudou a controlar parâmetros como o tamanho e a esferoidicidade, melhorando também a viabilidade celular. A influência de diferentes composições do meio de cultura na atividade metabólica celular e na diferenciação condrogénica foi avaliada em culturas celulares 2D. A adição de TGF-β1, bem como de outros suplementos, ao meio condrogénico (CM) aumentou a proliferação celular, e induziu a condrogénese, tendo sido revelada através de coloração histoquímica. Para a avaliação do processo condrogénico em modelos tridimensionais, os esferóides foram inicialmente preparados e maturados durante 14 dias (tendo-se adicionado 50 ng/mL de CMC ao meio de cultura no quinto dia) em placas ULA (revestidas com poly-HEMA) em BM com elevado teor de glicose (4.5 g/L), e com uma alta densidade celular. Depois, um grupo esferóides foi exposto ao CM; e outro mantido em BM, como grupo de controlo (CTRL) durante mais 28 dias (indução da condrogénese). O crescimento dos esferóidesfoi observado no grupo CM, enquanto que a sua contração foi evidente no grupo CTRL. Além disso, o CM conduziu a uma viabilidade celular superior. A coloração histoquímica só foi bem sucedida utilizando o sistema Fast Green/Safranin O, revelando a ocorrência de condrogénese nos esferóides expostos ao CM. A atividade ALP revelou a ausência do fenótipo hipertrófico nos esferóides diferenciados (condroesferas). Finalmente, os ensaios ELISA permitiram a deteção de colagénio tipo II e agrecano nas mesmas

Engineering Beta-Cell Spheroids for Type 1 Diabetes Treatment

Diabetes mellitus, the third most common disease in the world, is a chronic metabolic disorder caused by a failure of insulin production and/or an inability to respond to insulin. Specifically, type 1 diabetes is a disorder characterized by targeted autoimmune-directed destruction of a patient\u27s beta-cell population within the pancreatic islets of Langerhans. The current primary treatment for type 1 diabetes is daily multiple insulin injections. However, this treatment cannot provide sustained physiological release, and the insulin amount is not finely tuned to glycemia. Pancreatic transplants or islet transplants would be the preferred treatment method but the lack of donor tissue and immunoincompatibility has been shown to be a roadblock to their widespread use. The objective of this project is to develop an effective strategy for the treatment of type 1 diabetes using beta-cells based replacement therapy. To improve the viability of transplanted beta-cells, one novel approach is to transplant optimal size range of beta-cell spheroids rather than cell suspension. Uniform sized multicellular spheroids can be coated with a thin layer of non-degradable hydrogel for immunoisolation. In addition, the survival of spheroids of optimized size can be further improved with a novel coating of multiple layers of human mesenchymal stem cells (hMSCs), a cell type that has profound immunoregulatory effect, to prevent graft rejection. To prevent hMSC migrate away from spheroids, another layer of non-degradable hydrogel can be added. To further improve the viability and suppress the immune rejection, spheroids will be encapsulated with nanoparticles loaded with angiogenic and immune regulatory molecules. By this means the spheroid will passively evade the complications of stressors in addition to actively modulating the immune microenvironment for regulatory tolerance and long-term engraftment. Firstly, through optimizing our hydrogel systems based on poly (ethylene glycol) (PEG), we have created specific niche for beta-cells to form artificial islets in vitro. We have found that the optimal condition is the concentration of PEG at 5% and the ratios of 4-arm thiolated PEG to 4-arm PEG acrylate at 1:2. Conjugated with adhesive peptides, especially, RGD at 0.2 mM, can significantly promote the glucose stimulated insulin secretion of encapsulated beta-cells. Secondly, we have fabricated different sizes of uniformed beta-cells spheroids through our designed high-throughput automatic spheroids maker. Beta-cells in the spheroids of 200 μm exhibited largest insulin secretion based on glucose stimulus when compared to others with sizes of 100, 300, 400 and 500 μm. The novel core-shell structured spheroids-hMSCs complex was successfully achieved. Methylcellulose hydrogel was applied as physical barrier on the surface of beta-cells spheroids to inhibit invasion of hMSCs. Human MSCs prevented apoptosis of beta-cells spheroids and benefited insulin secretion when exposed to pro-inflammatory cytokines. Thirdly, immune regulatory molecules [leukemia inhibitory factor (LIF) and interleukin 10 (IL-10)] and angiogenic molecule [vascular endothelial growth factor (VEGF)] loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been successfully fabricated through solvent extraction/evaporation technique. These growth factors can be controlled release about 6 weeks. The bioactivity of released VEGF has been confirmed by the in vitro HAEC proliferation assay. Finally, beta-cells spheroids were transplanted under the kidney capsule to treat diabetic mice. Beta-cells spheroids kept the glucose level of diabetic mice constant. Co-transplanted hMSCs suppressed the host inflammation response, activated the regulatory T cells and also promoted angiogenesis at the transplantation site. The beta-cells spheroids/hMSCs/hydrogel complex initiated a mild inflammatory response. The LIF and IL-10, and VEGF loaded complex can further inhibited this response and promoted blood vessel network formation at the transplantation site. Our approach holds a great potential to treat type 1 diabetes

Integrative methods for reconstruction of dynamic networks in chondrogenesis

Application of human mesenchymal stem cells represents a promising approach in the field of regenerative medicine. Specific stimulation can give rise to chondrocytes, osteocytes or adipocytes. Investigation of the underlying biological processes which induce the observed cellular differentiation is essential to efficiently generate specific tissues for therapeutic purposes. Upon treatment with diverse stimuli, gene expression levels of cultivated human mesenchymal stem cells were monitored using time series microarray experiments for the three lineages. Application of gene network inference is a common approach to identify the regulatory dependencies among a set of investigated genes. This thesis applies the NetGenerator V2.0 tool, which is capable to deal with multiple time series data, which investigates the effect of multiple external stimuli. The applied model is based on a system of linear ordinary differential equations, whose parameters are optimised to reproduce the given time series datasets. Several procedures in the inference process were adapted in this new version in order to allow for the integration of multiple datasets. Network inference was applied on in silico network examples as well as on multi-experiment microarray data of mesenchymal stem cells. The resulting chondrogenesis model was evaluated on the basis of several features including the model adaptation to the data, total number of connections, proportion of connections associated with prior knowledge and the model stability in a resampling procedure. Altogether, NetGenerator V2.0 has provided an automatic and efficient way to integrate experimental datasets and to enhance the interpretability and reliability of the resulting network. In a second chondrogenesis model, the miRNA and mRNA time series data were integrated for the purpose of network inference. One hypothesis of the model was verified by experiments, which demonstrated the negative effect of miR-524-5p on downstream genes

Exploring the improvement of human cell cryopreservation

Regenerative medicine is an emerging technology and with hundreds of cell therapies currently in clinical trials there is a need to expand the limited knowledge related to their storage, shipment and preservation. The most widely used medium for human cell cryopreservation is 10%wt dimethyl sulfoxide (DMSO) in serum. However given its potential toxicity, DMSO usage is a key issue in cryopreservation. Methods specify the need to reduce cell exposure time to DMSO above 0°C as much as possible but the maximum amount of time cells can be exposed to DMSO to prevent a detrimental effect needs to be clarified. There are also regulatory issues and concerns with the xenotoxicity, ethics and supply of the other core component in the standard cryomedia formulation: Foetal Bovine Serum (FBS). Developing a viable alternative to FBS is crucial. In cryobiology literature thawing appears poorly understood. A stable process is as vital as freezing to prevent injury to cells. Protocols are currently too vague for cell therapy regulation and need improvement. The time dependent DMSO cytotoxicity was evaluated by overexposing cells to DMSO during and/or after cryopreservation. A broad investigation found that after 1 hour overexposure post thaw viability of human mesenchymal stem cells (hMSCs) was reduced from 96.3±0.6% to 74.1±4.0% and the co-expression of five key hMSC markers was changed from 97.9±1.3% to 68.3±2.6%. This significant change could cause indicate a change in product efficacy and affect patient health, to prevent this, DMSO exposure must be kept to below 1 hour. A range of alternative vehicle solutions were screened and human platelet lysate (hPL) investigated as an alternative. In depth experimentation with hPL as a cryopreservation vehicle solution and culture supplement (in place of FBS) found it to be a worthy, statistically similar alternative. With no xenological or ethical concerns, lower costs than other serum-free alternatives hPL could allow for a move away from xenological components. A heat transfer model was developed and determined that 720J is required to thaw a vial. Using the heat transfer model and additional factors such as pre-thaw stabilisation and on thaw dilution, a two-stage experiment found that the current standard process (warming in a 37°C waterbath) within the current paradigm of a 1.8mL cryovial is optimal but further work is required to define the process for scaled-up product

Device for Evaluating the Contraction of Cardiac Cell-Seeded Fibers

Drug-induced cardiotoxicity remains a primary reason why new pharmaceutical compounds are withdrawn from clinical trials and the consumer market. We have developed a novel cardiotoxicity screening device that utilizes cardiomyocytes seeded on fibrin microthreads to form a three-dimensional model for cardiac tissue. Our device can be used to analyze the contractile properties of these seeded threads. Upon the addition of pharmaceutical agents, the device has the potential to detect changes in contraction and thus identify cardiotoxic compounds