Automation, Monitoring, and Standardization of Cell Product Manufacturing.

Although regenerative medicine products are at the forefront of scientific research, technological innovation, and clinical translation, their reproducibility and large-scale production are compromised by automation, monitoring, and standardization issues. To overcome these limitations, new technologies at software (e.g., algorithms and artificial intelligence models, combined with imaging software and machine learning techniques) and hardware (e.g., automated liquid handling, automated cell expansion bioreactor systems, automated colony-forming unit counting and characterization units, and scalable cell culture plates) level are under intense investigation. Automation, monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside, reducing healthcare expenditure and improving services and patient care.

Recent Progress in European Advanced Therapy Medicinal Products and Beyond.

Cell- and gene-based therapies form one of the pillars of regenerative medicine. They have the potential to transform quality of life and improve the health status of patients with genetic and cellular defects, including genetic diseases, neurodegenerative diseases and tissue malignancies, amongst others. Despite numerous challenges, in the last decade, tremendous unified efforts by research and clinical scientists in academic, translational and industry settings have resulted in tangible outcomes in the form of many marketing authorizations and approved commercial firsts, such as Glybera®, Kymriah®, YESCARTA®, Holoclar®, and Luxturna™. This report presents a succinct analysis of developments in the regenerative medicine landscape, including immuno-oncology, with a focus on the European Union and examples of clinical and commercial successes and failures. The factors that led to these exciting developments in immune-oncology are also considered. Concurrently, several key issues, spanning from the identification of unmet clinical need, associated challenges, economic evaluation to policy improvements are emphasized. Furthermore, industry insights encompassing the five-dimensional research and development framework for the focused development of medicine, pricing and reimbursement issues, technology adoption and permeation of innovative advanced therapy medicinal products in the clinical set up are reflected upon, following elaborate discussions that transpired in different thematic tracks of Tissue Engineering & Regenerative Medicine International Society European Chapter 2017 Industry Symposium.

Evaluating the Past, Present, and Future of Regenerative Medicine: A Global View.

"Evaluating the Past and Present of Regenerative Medicine (RM)" was the first part of an Industry Symposium dedicated to the subject during the 2015 TERMIS World Congress in Boston. This working session presented a critical review of the current RM landscape in Europe and North America with possible projections for the future. Interestingly, the RM development cycle seems to obey the Gartner hype cycle, now at the enlightenment phase, after past exaggerated expectations and discouragements, as suggested by increasing numbers of clinical trials and recent market approvals of RM solutions in both Europe (Glybera and Holoclar® from Chiesi Pharma and Strimvelis® from GSK) and Japan (Remestemcel-L from Mesoblast®). The successful commercial translation of RM research is governed by five major drivers: (i) fully validated manufacturing capability for autologous or allogeneic products, (ii) reimbursement for targeted clinical indications with high and demonstrable medico-economic benefits versus standard of care, (iii) implication of regulatory bodies in the design and development plan of any RM solution, which should be well characterized, robust, with proven consistent efficacy and an acceptable and controlled positive benefit/risk ratio, (iv) collaborations facilitated by multicompetence hubs/consortia of excellence, (v) well-thought-out clinical development plans for reducing the risk of failure. Benefiting from past and present experience, the RM burgeoning industry is expected to accelerate the market release of cost-effective RM products with real curative potential for specific clinical indications with high unmet needs. This should be achieved by wisely leveraging all possible synergies of the different stakeholders, for example, patients, clinicians, reimbursement and health technology assessment (HTA) agencies, regulatory authorities, public/private investors, academia, and companies.

How Regenerative Medicine Stakeholders Adapt to Ever-Changing Technology and Regulatory Challenges? Snapshots from the World TERMIS Industry Symposium (September 10, 2015, Boston).

Regenerative medicine (RM) is a fascinating area of research and innovation. The huge potential of the field has been fairly underexploited so far. Both TERMIS-AM and TERMIS-EU Industry Committees are committed to mentoring and training young entrepreneurs for more successful commercial translation of upstream research. With this objective in mind, the two entities jointly organized an industry symposium during the past TERMIS World Congress (Boston, September 8-11, 2015) and invited senior managers of the RM industry for lectures and panel discussions. One of the two sessions of the symposium-How to overcome obstacles encountered when bringing products to the commercial phase?-aimed to share the inside, real experiences of leaders from TEI Biosciences (an Integra Company), Vericel (formerly Aastrom; acquirer of Genzyme Regenerative Medicine assets), RegenMedTX (formerly Tengion), Mindset Rx, ViThera Pharmaceuticals, and L'Oreal Research & Innovation. The symposium provided practical recommendations for RM product development, for remaining critical and objective when reviewing progress, for keeping solutions simple, and for remaining relevant and persistent.

Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFß-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50-100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60-100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Turning Regenerative Medicine Breakthrough Ideas and Innovations into Commercial Products.

The TERMIS-Europe (EU) Industry committee intended to address the two main critical issues in the clinical/commercial translation of Advanced Therapeutic Medicine Products (ATMP): (1) entrepreneurial exploitation of breakthrough ideas and innovations, and (2) regulatory market approval. Since January 2012, more than 12,000 publications related to regenerative medicine and tissue engineering have been accepted for publications, reflecting the intense academic research activity in this field. The TERMIS-EU 2014 Industry Symposium provided a reflection on the management of innovation and technological breakthroughs in biotechnology first proposed to contextualize the key development milestones and constraints of allocation of financial resources, in the development life-cycle of radical innovation projects. This was illustrated with the biofuels story, sharing similarities with regenerative medicine. The transition was then ensured by an overview of the key identified challenges facing the commercialization of cell therapy products as ATMP examples. Real cases and testimonies were then provided by a palette of medical technologies and regenerative medicine companies from their commercial development of cell and gene therapy products. Although the commercial development of ATMP is still at the proof-of-concept stage due to technology risks, changing policies, changing markets, and management changes, the sector is highly dynamic with a number of explored therapeutic approaches, developed by using a large diversity of business models, both proposed by the experience, pitfalls, and successes of regenerative medicine pioneers, and adapted to the constraint resource allocation and environment in radical innovation projects.

Translating cell-based regenerative medicines from research to successful products: challenges and solutions.

The Tissue Engineering & Regenerative Medicine International Society-Europe (TERMIS-EU) Industry Committee as well as its TERMIS-Americas (AM) counterpart intend to address the specific challenges and needs facing the industry in translating academic research into commercial products. Over the last 3 years, the TERMIS-EU Industry Committee has worked with commercial bodies to deliver programs that encourage academics to liaise with industry in proactive collaborations. The TERMIS-EU 2013 Industry Symposium aimed to build on this commercial agenda by focusing on two topics: Operations Management (How to move a process into the good manufacturing practice [GMP] environment) and Clinical Translation (Moving a GMP process into robust trials). These topics were introduced by providing the synergistic business perspective of partnering between the multiple regenerative medicine stakeholders, throughout the life cycle of product development. Seven industry leaders were invited to share their experience, expertise, and strategies. Due to the complex nature of regenerative medicine products, partnering for their successful commercial development seems inevitable to overcome all obstacles by sharing experiences and expertise of all stakeholders. When ideally implemented, the "innovation quotient" of a virtual team resulting from the combination of internal and external project teams can be maximized through maximizing the three main dimensions: core competences, technology portfolio, and alliance management.

Biochemical and biomechanical characterization of porcine small intestinal submucosa (SIS): a mini review.

Porcine small intestinal submucosa (SIS [Oasis(®)]) is an acellular, biological extracellular matrix (ECM) that has been found to significantly improve the healing of difficult-to-heal or chronic wounds in humans. Like dermal ECM, SIS contains collagen, elastin, glycosaminoglycans, proteoglycans, and growth factors that play important roles in healing. Preclinical studies have shown that numerous cell types attach to SIS, proliferate and migrate into the matrix, and differentiate. In addition, SIS can reduce the activity of matrix metalloproteinases (MMPs)-endogenous proteolytic enzymes whose levels and activities are increased in chronic wounds. Compared to the original single-layer SIS, multi-layer SIS has stronger mechanical properties and is more slowly degraded in wounds. Together, these SIS products provide flexibility in the selection of biologically-active ECMs that may be useful for the repair of diverse wound types.

Epidermal stem cells are preserved during commercial-scale manufacture of a bilayered, living cellular construct (Apligraf®).

It is unknown if epidermal stem cells are maintained during the commercial-scale manufacture of Apligraf, a bilayered living cellular construct (BLCC). To answer this question, we genetically marked replicating keratinocytes, derived from production-scale expansion of working cell banks, in two-dimensional culture with a beta-galactosidase-expressing retrovirus and monitored their fate after incorporation into BLCC and subsequent in vivo transplantation to a nude mouse. Histological analysis of BLCCs showed distinct beta-galactosidase-positive clusters similar to clonal proliferation units visible 8-32 weeks after grafting. Keratinocytes recovered from grafts at week 32 were expanded in vitro in two-dimensional culture, and clonal growth of recovered cells was then compared to the original pregraft population of keratinocytes by colony-forming efficiency (CFE) assays. The CFE of the cells regrown from the grafts was similar to pregraft CFEs (45% and 40%, respectively). Cells regrown from the grafts were then used to produce a second BLCC and generated a well-differentiated epithelium that was histologically similar to pregraft BLCC. These findings provide clear evidence that epidermal stem cells were sustained during the process of large-scale tissue fabrication and that the process of isolation and expansion of cells in BLCC construction retains viable stem cells.

De novo synthesis of human dermis in vitro in the absence of a three-dimensional scaffold.

Neonatal human dermal fibroblasts cultured in vitro synthesize an organized and physically substantial three-dimensional extracellular matrix, without the addition of exogenous matrix components or synthetic scaffolds. De novo matrix synthesis proceeds in an orderly manner over a 21-d culture period and beyond. Analysis of the fibroblast phenotype, i.e., matrix synthesis by the fibroblasts, suggests that both serum and serum-free conditions are conducive to the production of a human tissue-engineered "dermal equivalent". We report that given the appropriate permissive environment, the fibroblasts establish and grow a tissue in vitro, which bears striking biochemical and physical resemblance to normal human dermis.

Combined use of a collagen-based dermal substitute and a fibrin-based cultured epithelium: a step toward a total skin replacement for acute wounds.

Integra, a dermal replacement, is used as an immediate and temporary coverage for acute wounds, after which, autograft is used to reconstitute permanently the epidermal coverage. The fibrin sheet-cultured epithelium autograft (FS-CEA) could provide an effective alternative to the surgical procedure. To evaluate this hypothesis, we compared the association of Integra/FS-CE to Integra/control-cultured epithelium (control-CE). Their respective abilities: (1) to produce dermal-epidermal construct in vitro; (2) to generate skin replacement when grafted onto athymic mice were studied. We have shown that: (1) 83% of the FS-CE attached to the artificial dermis in vitro compared to only 33% for control-CE; (2) retraction of the grafted area was significantly lower 2 weeks after grafted with FS-CE than with the control-CE (P < 0.05); (3) 83% of the mice grafted with FS-CE showed the presence of a differentiated human epidermis 21 days after grafting, while such an epidermis was absent in all the animals of the control-CE group. We found that the use of FS-CE greatly improved adhesion, development of the epithelium and graft take onto the artificial dermis. We believe this technology should significantly improve the performance of dermal-epidermal skin replacement for acute wounds.

Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix.

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.