Concise review: mind the gap: challenges in characterizing and quantifying cell- and tissue-based therapies for clinical translation.

There are many challenges associated with characterizing and quantifying cells for use in cell- and tissue-based therapies. From a regulatory perspective, these advanced treatments must not only be safe and effective but also be made by high-quality manufacturing processes that allow for on-time delivery of viable products. Although sterility assays can be adapted from conventional bioprocessing, cell- and tissue-based therapies require more stringent safety assessments, especially in relation to use of animal products, immune reaction, and potential instability due to extended culture times. Furthermore, cell manufacturers who plan to use human embryonic stem cells in their therapies need to be particularly stringent in their final purification steps, due to the unrestricted growth potential of these cells. This review summarizes the current issues in characterization and quantification for cell- and tissue-based therapies, dividing these challenges into the regulatory themes of safety, potency, and manufacturing quality. It outlines current assays in use, as well as highlights the limits of many of these product release tests. Mode of action is discussed, with particular reference to in vitro surrogate assays that can be used to provide information to correlate with proposed in vivo patient efficacy. Importantly, this review highlights the requirement for basic research to improve current knowledge on the in vivo fate of these treatments; as well as an improved stakeholder negotiation process to identify the measurement requirements that will ensure the manufacture of the best possible cell- and tissue-based therapies within the shortest timeframe for the most patient benefit.

Human pilot studies reveal the potential of a vitronectin: growth factor complex as a treatment for chronic wounds.

Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.

Finding the culprit: a review of the influences of proteases on the chronic wound environment.

Chronic leg ulcers are a complex medical condition with varied underlying causes and requiring diverse treatment strategies. It is generally accepted that chronic ulcers occur when the normal wound healing process is interrupted. These wounds are characterized by excessive protease activity, abundant granulation tissue, and decreased levels of growth factors, resulting in an overall poor prognosis for the patient. Many studies have focused on identifying the key proteases, specifically matrix metalloproteinases (MMPs), responsible for an ulcer's chronicity. Of note, the results of these studies are often conflicting. This report therefore focuses on a review of this literature to identify which MMPs are important in terms of ulcer prognosis and healing outcome. This has revealed that MMPs are clearly important in many biological processes in wound healing, hence are critical to consider when developing improved therapies to enhance both ulcer healing times and ulcer healing outcomes.

Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalised hydrogels.

Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.

Qualification of academic facilities for small-scale automated manufacture of autologous cell-based products.

Academic centers, hospitals and small companies, as typical development settings for UK regenerative medicine assets, are significant contributors to the development of autologous cell-based therapies. Often lacking the appropriate funding, quality assurance heritage or specialist regulatory expertise, qualifying aseptic cell processing facilities for GMP compliance is a significant challenge. The qualification of a new Cell Therapy Manufacturing Facility with automated processing capability, the first of its kind in a UK academic setting, provides a unique demonstrator for the qualification of small-scale, automated facilities for GMP-compliant manufacture of autologous cell-based products in these settings. This paper shares our experiences in qualifying the Cell Therapy Manufacturing Facility, focusing on our approach to streamlining the qualification effort, the challenges, project delays and inefficiencies we encountered, and the subsequent lessons learned.

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

AIM: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. MATERIALS & METHODS: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. RESULTS & CONCLUSION: Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Precision manufacturing for clinical-quality regenerative medicines.

Innovations in engineering applied to healthcare make a significant difference to people's lives. Market growth is guaranteed by demographics. Regulation and requirements for good manufacturing practice-extreme levels of repeatability and reliability-demand high-precision process and measurement solutions. Emerging technologies using living biological materials add complexity. This paper presents some results of work demonstrating the precision automated manufacture of living materials, particularly the expansion of populations of human stem cells for therapeutic use as regenerative medicines. The paper also describes quality engineering techniques for precision process design and improvement, and identifies the requirements for manufacturing technology and measurement systems evolution for such therapies.

Regenerative medicine, resource and regulation: lessons learned from the remedi project.

The successful commercialization of regenerative medicine products provides a unique challenge to the manufacturer owing to a lack of suitable investment/business models and a constantly evolving regulatory framework. The resultant slow translation of scientific discovery into safe and clinically efficacious therapies is preventing many potential products from reaching the market. This is despite of the need for new therapies that may reduce the burden on the world's healthcare systems and address the desperate need for replacement tissues and organs. The collaborative Engineering and Physical Sciences Research Council (EPSRC)-funded remedi project was devised to take a holistic but manufacturing-led approach to the challenge of translational regenerative medicine in the UK. Through strategic collaborations and discussions with industry and other academic partners, many of the positive and negative issues surrounding business and regulatory success have been documented to provide a remedi-led perspective on the management of risk in business and the elucidation of the regulatory pathways, and how the two are inherently linked. This article represents the findings from these discussions with key stakeholders and the research into best business and regulatory practices.