Commercialization of skin substitutes for third-degree burn wounds.

Technological advances have increasingly provided more and better treatment options for patients with severe burns. Here, we provide a bird's-eye view of the product development process for third-degree burn wounds with considerations of the critical interaction with regulatory bodies, existing technological gaps, and future directions for skin substitutes.

In-situ scalable manufacturing of Epstein-Barr virus-specific T-cells using bioreactor with an expandable culture area (BECA).

The ex-vivo expansion of antigen-specific T-cells for adoptive T-cell immunotherapy requires active interaction between T-cells and antigen-presenting cells therefore culture density and environment become important variables to control. Maintenance of culture density in a static environment is traditionally performed by the expansion of the culture area through splitting of culture from a single vessel into multiple vessels-a highly laborious process. This study aims to validate the use and efficacy of a novel bioreactor, bioreactor with an expandable culture area-dual chamber (BECA-D), that was designed and developed with a cell chamber with expandable culture area (12-108 cm2) and a separate media chamber to allow for in-situ scaling of culture with maintenance of optimum culture density and improved nutrient and gas exchange while minimizing disturbance to the culture. The performance of BECA-D in the culture of Epstein-Barr virus-specific T-cells (EBVSTs) was compared to the 24-well plate. BECA-D had 0.9-9.7 times the average culture yield of the 24-well plates across 5 donor sets. BECA-D was able to maintain the culture environment with relatively stable glucose and lactate levels as the culture expanded. This study concludes that BECA-D can support the culture of ex-vivo EBVSTs with lower manufacturing labour and time requirements compared to the use of the 24-well plate. BECA-D and its adaptation into a closed system with an automated platform (currently being developed) provides cell therapy manufacturers and developers with a closed scale-out solution to producing adoptive cell therapy for clinical use.

Decellularization systems and devices: State-of-the-art.

Extracellular matrix (ECM) is a natural biomaterial scaffold that provides biochemical and structural support to its surrounding cells, forming tissue and respective organs. These ECM proteins can be extracted from organs and tissues through decellularization, which is the process of removing cellular content and nuclear material from the organs to obtain decellularized ECM (dECM). dECM is a versatile and functional biomaterial that can be used as the base component of bioinks for rebuilding tissue and organs. Intact dECM of whole organs can be used as a scaffold for recellularization with human stem cells to produce a functioning organ. As decellularization is a relatively new lab process, the associated technologies and devices are largely non-standardized and only available in small, lab-specific scales. Additionally, there is a lack of standardized protocols to analyze the quality and consistency of harvested dECM for medical applications. This review discusses the relevant decellularization systems and devices currently available to facilitate further development of this process for larger scales with the intention to commercialize dECM materials. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) is a natural cocktail of biomaterials that provides biochemical and structural support to its surrounding cells. ECM proteins are extracted from organs and tissues through decellularization. Being a versatile and functional biomaterial, decellularized extracellular matrix (dECM) is being used as base component of bioinks/hydrogels for rebuilding of tissue and organ constructs. Decellularization is a relatively new lab process with associated technologies/devices being largely non-standardized and only available in lab-specific scales. We discuss categories of decellularization systems and devices for the first time being used in academic and commercial settings. We highlight inherent challenges with the current systems and suggest possible solutions. We comment on further development of these processes for large-scale and commercial applications of dECM.

Additive Biomanufacturing with Collagen Inks.

Collagen is a natural polymer found abundantly in the extracellular matrix (ECM). It is easily extracted from a variety of sources and exhibits excellent biological properties such as biocompatibility and weak antigenicity. Additionally, different processes allow control of physical and chemical properties such as mechanical stiffness, viscosity and biodegradability. Moreover, various additive biomanufacturing technology has enabled layer-by-layer construction of complex structures to support biological function. Additive biomanufacturing has expanded the use of collagen biomaterial in various regenerative medicine and disease modelling application (e.g., skin, bone and cornea). Currently, regulatory hurdles in translating collagen biomaterials still remain. Additive biomanufacturing may help to overcome such hurdles commercializing collagen biomaterials and fulfill its potential for biomedicine.

Commercialization of Organoids.

Organoids have been successfully exploited for drug screening, disease modeling, pathogenesis, and regenerative medicine. Herein, we discuss the progress achieved in the commercialization of organoids in the last few years. We further elaborate on the concept of organoid biobank and highlight ethical and regulatory issues surrounding organoid research and commercialization.

In situ bioprinting - Bioprinting from benchside to bedside?

Bioprinting technologies have been advancing at the convergence of automation, digitalization, and new tissue engineering (TE) approaches. In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when de novo tissues are to be printed directly on the intended anatomical location in the living body. To date, few attempts have been made to fabricate in situ tissues, which can be safely arrested and immobilized while printing in preclinical living models. In this review, we have explained the need and utility for in situ bioprinting with regard to the conventional bioprinting approach. The two main in situ bioprinting approaches, namely, robotic arm and handheld approaches, have been defined and differentiated. The various studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. Finally, we have also discussed the advantages, challenges, and the prospects in the field of in situ bioprinting modalities in line with parallel technological advancements. STATEMENT OF SIGNIFICANCE: In situ bioprinting may be favored during certain situations when compared with the conventional in vitro bioprinting when tissues are to be fabricated or repaired directly on the intended anatomical location in the living body, using the body as a bioreactor. However, the technology requires a lot more improvement to fabricate complex tissues in situ, which could eventually be possible through the multi-disciplinary innovations in tissue engineering. This review explains the need and utility and current approaches by handheld and robotic modes for in situ bioprinting. The latest studies involving in situ fabrication of skin, bone, and cartilage tissues have been elucidated. The review also covers the background studies, advantages, technical and ethical challenges, and possible suggestions for future improvements.

Healing of Chronic Wounds: An Update of Recent Developments and Future Possibilities.

Chronic wounds are the result of disruptions in the body's usual process of healing. They are not only a source of significant pain and discomfort but also, more importantly, an unguarded port of entry for pathogens into the body. While our current understanding of this phenomenon is far from complete, findings in physiological patterns and advancements in wound healing technologies have helped develop wound management and healing solutions to this long-standing medical challenge. This review presents an overview of known wound healing mechanics, abnormalities that lead to chronic wounds, and a summary of established and new wound healing technologies. Various approaches to heal wounds are discussed, from dermal replacements to advanced biomaterial-based treatments, from cell-, synthetic-, and composite-based approaches to preclinical approaches, which make developing such products possible. While tested breakthrough products are described, the authors focused more on recently developed innovations, which are at varying stages of maturity. The review concludes with a note on future perspectives and opinions on where the field and industry are headed and where they should be. Impact Statement Wound healing is an important area of research and clinical practice, and has captured the attention of tissue engineers since the nascent beginnings of the discipline. Tissue-engineered skin was the first FDA-approved product, achieved in 1996. Despite this success, and the passage of time, healing wounds, particularly chronic wounds, remains a vexing challenge. This comprehensive review article will provide readers with a synopsis of current issues, research approaches, animal models, technologies, and products that span the continuum from early development to clinical studies, in the hope of fueling new interests and ideas to overcome this long-standing medical challenge.

Inertial-Based Filtration Method for Removal of Microcarriers from Mesenchymal Stem Cell Suspensions.

Rapidly evolving cell-based therapies towards clinical trials demand alternative approaches for efficient expansion of adherent cell types such as human mesenchymal stem cells (hMSCs). Using microcarriers (100-300 µm) in a stirred tank bioreactor offers considerably enhanced surface to volume ratio of culture environment. However, downstream purification of the harvested cell product needs to be addressed carefully due to distinctive features and fragility of these cell products. This work demonstrates a novel alternative approach which utilizes inertial focusing to separate microcarriers (MCs) from the final cell suspension. First, we systematically investigated MC focusing dynamics inside scaled-up curved channels with trapezoidal and rectangular cross-sections. A trapezoidal spiral channel with ultra-low-slope (Tan(α) = 0.0375) was found to contribute to strong MC focusing (~300 < Re < ~400) while managing high MC volume fractions up to ~1.68%. Accordingly, the high-throughput trapezoidal spiral channel successfully separated MCs from hMSC suspension with total cell yield~94% (after two passes) at a high volumetric flow rate of ~30 mL/min (Re~326.5).

Organ-Derived Decellularized Extracellular Matrix: A Game Changer for Bioink Manufacturing?

The extracellular matrix (ECM) comprises a complex milieu of proteins and other growth factors that provide mechanical, biophysical, and biochemical cues to cells. The ECM is organ specific, and its detailed composition varies across organs. Bioinks are material formulations and biological molecules or cells processed during a bioprinting process. Organ-derived decellularized ECM (dECM) bioinks have emerged as arguably the most biomimetic bioinks. Here, we review bioinks derived from different decellularized organs, the techniques used to obtain these bioinks, and the characterization methods used to evaluate their quality. We emphasize that obtaining a good-quality bioink depends on the choice of organ, animal, and decellularization method. Finally, we explore potential large-scale applications of bioinks and challenges in manufacturing such bioinks.

Applying macromolecular crowding to 3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs.

Native tissues and/or organs possess complex hierarchical porous structures that confer highly-specific cellular functions. Despite advances in fabrication processes, it is still very challenging to emulate the hierarchical porous collagen architecture found in most native tissues. Hence, the ability to recreate such hierarchical porous structures would result in biomimetic tissue-engineered constructs. Here, a single-step drop-on-demand (DOD) bioprinting strategy is proposed to fabricate hierarchical porous collagen-based hydrogels. Printable macromolecule-based bio-inks (polyvinylpyrrolidone, PVP) have been developed and printed in a DOD manner to manipulate the porosity within the multi-layered collagen-based hydrogels by altering the collagen fibrillogenesis process. The experimental results have indicated that hierarchical porous collagen structures could be achieved by controlling the number of macromolecule-based bio-ink droplets printed on each printed collagen layer. This facile single-step bioprinting process could be useful for the structural design of collagen-based hydrogels for various tissue engineering applications.

Proof-of-concept: 3D bioprinting of pigmented human skin constructs.

Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.

The arrival of commercial bioprinters - Towards 3D bioprinting revolution!

The dawn of commercial bioprinting is rapidly advancing the tissue engineering field. In the past few years, new bioprinting approaches as well as novel bioinks formulations have emerged, enabling biological research groups to demonstrate the use of such technology to fabricate functional and relevant tissue models. In recent years, several companies have launched bioprinters pushing for early adoption and democratisation of bioprinting. This article reviews the progress in commercial bioprinting since the inception, with a particular focus on the comparison of different available printing technologies and important features of the individual technologies as well as various existing applications. Various challenges and potential design considerations for next generations of bioprinters are also discussed.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing.

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%-3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Skin Bioprinting: Impending Reality or Fantasy?

Bioprinting provides a fully automated and advanced platform that facilitates the simultaneous and highly specific deposition of multiple types of skin cells and biomaterials, a process that is lacking in conventional skin tissue-engineering approaches. Here, we provide a realistic, current overview of skin bioprinting, distinguishing facts from myths. We present an in-depth analysis of both current skin bioprinting works and the cellular and matrix components of native human skin. We also highlight current limitations and achievements, followed by design considerations and a future outlook for skin bioprinting. The potential of bioprinting with converging opportunities in biology, material, and computational design will eventually facilitate the fabrication of improved tissue-engineered (TE) skin constructs, making bioprinting skin an impending reality.

Improving umbilical cord blood processing to increase total nucleated cell count yield and reduce cord input wastage by managing the consequences of input variation.

BACKGROUND AIMS: With the rising use of umbilical cord blood (UCB) as an alternative source of hematopoietic stem cells, storage inventories of UCB have grown, giving rise to genetically diverse inventories globally. In the absence of reliable markers such as CD34 or counts of colony-forming units, total nucleated cell (TNC) counts are often used as an indicator of potency, and transplant centers worldwide often select units with the largest counts of TNC. As a result, cord blood banks are driven to increase the quality of stored inventories by increasing the TNC count of products stored. However, these banks face challenges in recovering consistent levels of TNC with the use of the standard protocols of automated umbilical cord processing systems, particularly in the presence of input variation both of cord blood volume and TNC count, in which it is currently not possible to process larger but useable UCB units with consequent losses in TNC. METHODS: This report addresses the challenge of recovering consistently high TNC yields in volume reduction by proposing and validating an alternative protocol capable of processing a larger range of units more reliably. RESULTS: This work demonstrates improvements in plastic ware and tubing sets and in the recovery process protocol with consequent productivity gains in TNC yield and a reduction in standard deviation. CONCLUSIONS: This work could pave the way for cord blood banks to improve UCB processing and increase efficiency through higher yields and lower costs.

Current status and perspectives on stem cell-based therapies undergoing clinical trials for regenerative medicine: case studies.

BACKGROUND: Apart from haematopoietic stem cell transplantation for haematological disorders many stem cell-based therapies are experimental. However, with only 12 years between human embryonic stem cell isolation and the first clinical trial, development of stem cell products for regenerative medicine has been rapid and numerous clinical trials have begun to investigate their therapeutic potential. SOURCE OF DATA: This review summarizes key clinical trial data, current and future perspectives on stem cell-based products undergoing clinical trials, based on literature search and author research. AREAS OF AGREEMENT: It is widely recognized that the ability to stimulate stem cell differentiation into specialized cells for use as cellular therapies will revolutionize health care and offer major hope for numerous diseases for which there are limited or no therapeutic options. AREAS OF CONTROVERSY: Stem cell-based products are unique and cover a large range of disorders to be treated; therefore, there is significant potential for variation in cell source, type, processing manipulation, the bioprocessing approach and scalability, the cost and purity of manufacture, final product quality and mode of action. As such there are gaps in regulatory and manufacturing frameworks and technologies, only a small number of products are currently within late phase clinical trials and few products have achieved commercialization. GROWING POINTS: Recent developments are encouraging acceleration through the difficulties encountered en route to clinical trials and commercialization of stem cell therapies. AREAS TIMELY FOR DEVELOPING RESEARCH: The field is growing year on year with the first clinical trial using induced pluripotent stem cells anticipated by end 2013.

Automatic algorithm for generating complex polyhedral scaffold structures for tissue engineering.

In this article, an approach for tissue-engineering (TE) scaffold fabrication by way of integrating computer-based medical imaging, computer graphics, data manipulation techniques, computer-aided design (CAD), and rapid prototyping (RP) technologies is introduced. The aim is to provide a generic solution for the production of scaffolds that can potentially meet the diverse requirements of TE applications. In the work presented, a novel parametric library of open polyhedral unit cells is developed to assist the user in designing the microarchitecture of the scaffold according to the requirements of its final TE application. Once an open polyhedral unit cell design is selected and sized, a specially developed algorithm is employed to assemble the microarchitecture of the scaffold while adhering to the external geometry of the patient's anatomy generated from medical imaging data. RP fabrication techniques are then employed to build the scaffolds according to the CAD-generated designs. The combined application of such technologies promises unprecedented scaffold qualities with spatially and anatomically accurate three-dimensional forms as well as highly consistent and reproducible microarchitectures. The integrated system also has great potential in providing new cost-effective and rapid solutions to customized made-to-order TE scaffold production.