Exosomes For Regenerative Medicine – Manufacturing Challenges And Potential Applications

Extracellular vesicles, or exosomes, are emerging as a novel class of therapeutic that confer many of the regenerative functions originally ascribed to the cells that produce them. From their emergence as possible biomarkers of diseases such as cancer they have rapidly emerged as candidate regenerative medicines. The potential market for exosomes is huge, spanning a range of clinical indications that stem cells have been expected to be utilized for. Overwhelmingly positive pre-clinical data on the effectiveness of exosomes has underpinned this excitement, yet there remain some substantial manufacturing challenges that relate to scale of production and reliability in measurement and characterization of exosome material. In particular, challenges of purifying exosomes at scale whilst preserving their therapeutic attributes need to be overcome to ensure products can be made consistently. The talk will introduce exosomes and their potential applications in regenerative medicine and consider the current manufacturing challenges that researchers are now starting to tackle

Virtual reality to rapidly scale a resilient workforce for cell and gene therapy manufacturing

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Towards an allogeneic therapy for neural regeneration

Injuries to the central nervous system (CNS) can be devastating. CNS injuries include those to the spinal cord, where there can be a complete loss of function below the point of injury. Spinal cord injuries impact up to 500,000 people worldwide every year and where function is lost, quality of life can be severely limited. Olfactory ensheathing cells (OECs) are a promising cell therapy candidate for treatment of neurologic injury as they have been shown to promote neuronal survival and facilitate regeneration of severed axons. Despite their unique properties, OECs are very challenging cells to work with since they are difficult to isolate, difficult to sustain in culture for prolonged periods and there is still controversy around how to characterize their identity and potency. Due to the inherent variability of OEC yield in biopsies and difficulties in growing these cells, producing an autologous therapy is not currently a viable option. Therefore, we sought to develop allogeniec OEC lines using a conditional immortalization tool. We undertook characterization work to ensure they expressed key putative OEC markers (such as p75NTR and S100β) and were able to support neuron extension in in vitro models. Subsequently, extensive bioprocess development was undertaken to investigate parameters such as: Cell culture conditions, e.g. effect of different culture media, initial seeding densities and microcarrier-based expansion Characterization process parameters, e.g. effect of culture time and pre-selection on identity marker expression, impact of detergents on identity marker measurement, and finally their impact in functional assays (including neuron co-culture to identify the impact of processing on neuronal support activity). A number of important insights have been gained from this work including: identity markers are transient making characterization challenging; and analytics methodologies employed themselves affect what is measurable. However, these insights are informing our future approach to creating candidate cell lines for treatment of neurologic injury

Microfluidic tools and high-content imaging for cell therapy bioprocessing

Culture parameters including type of matrix, feeding regimes and media formulations have to be fine-tuned in order to obtain the desired cell quality attributes that will be the basis of large scale bioprocess development. Costly and time consuming screenings, usually performed in multi-well plates, are undertaken to obtain preliminary results. In contrast, microfluidics offers the possibility to perform studies of different parameters in an integrated manner, with high-throughput analysis, lower consumption of reagents and reduction in time. This approach has the possibility to unveil valuable information in early stages of process development that can reduce the risks associated with scale-up. In one working example, a range of process parameters were screened for the development of a microcarrier-based cell culture of a commercial cell line. Two different media flow rates and four different microcarrier substrates were assessed for their capacity to sustain cell growth and known markers of different stages of cell differentiation. It was possible to define combinations of parameters that yield very distinct outcomes in terms of cell number and identity. In a second working example, modular production of bone-like tissue using mesenchymal stem cells, different implantable biomaterial scaffolds and different culture media feeds was assessed. In situ analysis identified optimal combinations of parameters that maintained multipotency in Mesenchymal Stem Cells (MSCs) and also combinations that promote MSCs maturation. Scaling up to 125 mL cultures enabled the production of large clusters of bone-like material

Impact of dual cell co-culture and cell-conditioned media on yield and function of a human olfactory cell line for regenerative medicine

Olfactory ensheathing cells (OECs) are a promising candidate therapy for neuronal tissue repair. However, appropriate priming conditions to drive a regenerative phenotype are yet to be determined. We first assessed the effect of using a human fibroblast feeder layer and fibroblast conditioned media on primary rat olfactory mucosal cells (OMCs). We found that OMCs cultured on fibroblast feeders had greater expression of the key OEC marker p75NTR (25.1 ± 10.7 cells/mm2) compared with OMCs cultured on laminin (4.0 ± 0.8 cells/mm2, p = 0.001). However, the addition of fibroblast-conditioned media (CM) resulted in a significant increase in Thy1.1 (45.9 ± 9.0 cells/mm2 versus 12.5 ± 2.5 cells/mm2 on laminin, p = 0.006), an undesirable cell marker as it is regarded to be a marker of contaminating fibroblasts. A direct comparison between human feeders and GMP cell line Ms3T3 was then undertaken. Ms3T3 cells supported similar p75NTR levels (10.7 ± 5.3 cells/mm2) with significantly reduced Thy1.1 expression (4.8 ± 2.1 cells/mm2). Ms3T3 cells were used as feeder layers for human OECs to determine whether observations made in the rat model were conserved. Examination of the OEC phenotype (S100β expression and neurite outgrowth from NG108-15 cells) revealed that co-culture with fibroblast feeders had a negative effect on human OECs, contrary to observations of rat OECs. CM negatively affected rat and human OECs equally. When the best and worst conditions in terms of supporting S100β expression were used in NG108-15 neuron co-cultures, those with the highest S100β expression resulted in longer and more numerous neurites (22.8 ± 2.4 μm neurite length/neuron for laminin) compared with the lowest S100β expression (17.9 ± 1.1 μm for Ms3T3 feeders with CM). In conclusion, this work revealed that neither dual co-culture nor fibroblast-conditioned media support the regenerative OEC phenotype. In our case, a preliminary rat model was not predictive of human cell responses

Investigating the requirement for dual cell co-culture platforms in creating regenerative cell therapies for CNS injury

Injuries to the central nervous system (CNS) can be devastating. CNS injuries include those to the spinal cord, where there can be a complete loss of function below the point of injury. Spinal cord injury impacts up to 500,000 people worldwide every year and where function is lost, quality of life can be severely limited. Olfactory ensheathing cells (OECs) are a candidate cell therapy for spinal cord injury as they can promote neuronal survival and facilitate regeneration of severed axons. Despite their unique properties, OECs are very challenging cells to work with because they are difficult to isolate, difficult to sustain in culture for prolonged periods and there is still controversy around how to characterize their identity and potency. The overall aim of this project is to identify methods to enhance the survival, growth and function of OECs. It has been reported that for OECs to be truly functional they require interaction with fibroblasts. Therefore, we sought to investigate whether it is necessary to use fibroblasts as a feeder layer to support OECs via physical cell-cell contact, or whether paracrine soluble factors in the conditioned media from fibroblasts would provide the trophic support necessary to enhance OEC survival and growth. A human mucosal fibroblast cell line was used as a feeder layer. Primary rat OECs were cultured for 14 days on the feeder layer or control substrate (laminin-coated dishes). After 14 days, the morphology of cells was assessed and an algorithm generated using ImageJ was used to ascribe a mathematical value to OEC morphology to determine whether a correlation of morphology to expression of markers could be made. Cells cultured on feeders adopted a more spindle-like appearance compared with cells cultured on laminin, which adopted an enlarged morphology. The algorithm was used to analyse the circularity of cells that labelled positive for candidate identity marker S100b. It was found that cells cultured on feeders had a lower circularity, and therefore more elongated shape compared to those cultured on laminin (p=0.037). Additionally, a significant increase in p75NTR expression (a second candidate OEC marker) was observed (p=0.01) on feeders. To further investigate the relationship between the OECs and the feeders, cells were cultured in the presence of conditioned media from the fibroblasts. When cells were cultured in conditioned media there was a significant (p=0.002) upregulation of Thy1, an undesirable marker, and the significance of this will be investigated further with work underway to compare different feeder types and their impact on marker expression

Development of downstream processing options for the commercial scale purification of stem cell derived exosomes

Exosomes (or extracellular vesicles) are rapidly gaining momentum as a novel type of regenerative medicine. Exosomes are nano-vesicles in the size range of 20-150nm that are secreted by mammalian cell types (including stem cells). They can induce potent biological functions in surrounding target cells to induce effects that mimic those of the producing cell. These functions can be regenerative, immunomodulatory, anti-scarring and even anti-cancer depending on the state of the producer cell. Consequently, there is enormous potential to develop a range of function-specific products across a wide variety of indications similar to macromolecular blockbusters, but which extend beyond symptom management to produce curative outcomes. However, current methods for purifying exosomes have been based on technologies grandfathered in from the viral industry which are not suitable for large scale, high purity production but are robust enough to perform preliminary characterisation of the exosome population composition. Please click Additional Files below to see the full abstract

Manufacturing Exosomes:A Promising Therapeutic Platform

Extracellular vesicles, in particular the subclass exosomes, are rapidly emerging as a novel therapeutic platform. However, currently very few clinical validation studies and no clearly defined manufacturing process exist. As exosomes progress towards the clinic for treatment of a vast array of diseases, it is important to define the engineering basis for their manufacture early in the development cycle to ensure they can be produced cost-effectively at the appropriate scale. We hypothesize that transitioning to defined manufacturing platforms will increase consistency of the exosome product and improve their clinical advancement as a new therapeutic tool. We present manufacturing technologies and strategies that are being implemented and consider their application for the transition from bench-scale to clinical production of exosomes

Microcarrier expansion of c-MycERTAM - modified human olfactory mucosa cells for neural regeneration

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 10 5 cells/ml and 4.9 × 10 4 cells/ml for plastic and plastic plus, respectively, after 7 days. p75 NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields