Application of a simple unstructured kinetic and cost of goods models to support T-cell therapy manufacture.

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation-based model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 106  cell h-1 ) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.

The Use of Platelet-Rich Plasma (PRP) for the Management of Non-union Fractures.

PURPOSE OF REVIEW: The treatment of non-union fractures represents a significant challenge for orthopaedic surgeons. In recent years, biologic agents have been investigated and utilised to support and improve bone healing. Among these agents, platelet-rich plasma (PRP) is an emerging strategy that is gaining popularity. The aim of this review is to evaluate the current literature regarding the application and clinical effectiveness of PRP injections, specifically for the treatment of non-union fractures. RECENT FINDINGS: The majority of published studies reported that PRP accelerated fracture healing; however, this evidence was predominantly level IV. The lack of randomised, clinical trials (level I-II evidence) is currently hampering the successful clinical translation of PRP as a therapy for non-union fractures. This is despite the positive reports regarding its potential to heal non-union fractures, when used in isolation or in combination with other forms of treatment. Future recommendations to facilitate clinical translation and acceptance of PRP as a therapy include the need to investigate the effects of administering higher volumes of PRP (i.e. 5-20 mL) along with the requirement for more prolonged (> 11 months) randomised clinical trials.

The importance of cell culture parameter standardization: an assessment of the robustness of the 2102Ep reference cell line.

Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing 'quick hit' experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results demonstrated that specific growth rates (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to medium exchange after 48h prior to reseeding at 72h (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (route A1); whereby SGR values were (0.021±0.004) and (0.019±0.004). Viability was higher in route B2 over 10 passages with average viability reported as (86.3%±8.1) compared to route A1 (83.3±8.8). The metabolite data demonstrated both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had consistent-specific metabolite rates (SMR) for glucose, but SMR values of route B1 was consistently lower than route B2 (0.00001 mmol, cell-1.d-1 and 0.000025).Results revealed interactions between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve culture for therapeutic use, biologics production, and reference lines.

Distributed automated manufacturing of pluripotent stem cell products.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site-decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing 'live' corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

Gene modification strategies using AO-mediated exon skipping and CRISPR/Cas9 as potential therapies for Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease affecting 1 in 5000 young males worldwide annually. Patients experience muscle weakness and loss of ambulation at an early age, with ∼75% reduced life expectancy. Recently developed genetic editing strategies aim to convert severe DMD phenotypes to a milder disease course. Among these, the antisense oligonucleotide (AO)-mediated exon skipping and the adeno-associated viral-delivered clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (adeno-associated viral (AAV)-delivered CRISPR/Cas9) gene editing have shown promising results in restoring dystrophin protein expression and functionality in skeletal and heart muscle in both animals and human cells in vivo and in vitro. However, therapeutic benefits currently remain unclear. The aim of this review is to compare the potential therapeutic benefits, efficacy, safety, and clinical progress of AO-mediated exon skipping and CRISPR/Cas9 gene-editing strategies. Both techniques have demonstrated therapeutic benefit and long-term efficacy in clinical trials. AAV-delivery of CRISPR/Cas9 may potentially correct disease-causing mutations following a single treatment compared to the required continuous AO/PMO-delivery of exon skipping drugs. The latter has the potential to increase the dystrophin expression in skeletal/heart muscle with sustained effects. However, therapeutic challenges including the need for optimised delivery must be overcome in to advance current clinical data.

Correction to: The efficacy of different sources of mesenchymal stem cells for the treatment of knee osteoarthritis.

There is an error in the Original Publication of this paper. The author names were incorrectly presented.

The efficacy of different sources of mesenchymal stem cells for the treatment of knee osteoarthritis.

Osteoarthritis (OA) is a common cause of chronic pain and disability. Regenerative therapies using mesenchymal stem cells (MSCs) provide an option for OA treatment as it could potentially regenerate the damaged cartilage. Bone marrow, adipose tissue and synovium are common MSC sources. The aim is to compare the therapeutic effect of MSCs from bone marrow, adipose tissue and synovium; combining its differentiation potential and accessibility, to decide the optimal source of MSCs for the treatment of knee OA. A comparison of preclinical and clinical studies using MSCs has been made with regard to treatment outcomes, isolation procedure and differentiation potential. All types of MSCs are effective at improving the clinical and structural condition of OA patients, but the longevity of the treatment, i.e. an effect that is maintained for at least 2 years, cannot be guaranteed. This review highlighted great variations in selection criteria and culture expansion conditions of MSCs between the literature and clinical trials. It also emphasised a substantial diversity and lack of consistency in the assessment mythology of clinical outcome after completion of MSC therapies procedures. A more cohesive methodology is required to evaluate the outcome of MSC treatments using quantitative and standardised frameworks in order to be able to directly compare results. Larger population of patients are recommended to assess the quality of MSC when designing studies and clinical trials to reaffirm the efficacy of MSC treatment prior to and within the clinical trials and follow up studies.