Approach of resource expenditure estimation toward mechanization in the manufacturing of cell-based products.

Recent developments for the manufacturing of cell-based products have focused on the advancement of products to clinical trials or commercialization, with awareness of the importance of cost-based effectiveness in cell manufacturing. The mechanization of cell-processing operations is advantageous for the reproducibility and stability of product quality and is thought to reduce the cost-of-goods through the life cycle of the product in a scale-up system; however, few cases of the implementation exist. This study developed an estimation method for the resource expenditure of cell-processing operations in the manufacturing of cell-based products. To estimate resource expenditures, we evaluated the manufacturing processes by operations involving entering into the surrounding area of cell processing zone, materials loading, cell-processing operation, cleaning, and leaving from the surrounding area. The cell-processing operation is applicable to manual or robotic cell manufacturing system in a biosafety cabinet or an isolator system. In cases of low annual batch numbers of manufacturing (batch number <33), the resource expenditure of cell-processing operations in a robotic operation system installed in the isolator system is estimated to be higher compared with a manual operation system in the isolator system due to additional initial costs for design and fabrication of the robotic operation system containing robot arms. With increasing numbers of annual batches, the resource expenditure decreases for robotic operating system, leading to an advantageous juncture where the resource expenditure of a robotic operation system is equivalent to that of a manually operated system, whereby the labor cost for cell-processing operations rises. In addition, the expertise of operations required for cell manufacturing is suggested to foster potential risks associated with the operation skills or turnover of operators, and the cost of education and training increases due to the necessity of persistent human resource development. Collectively, revealing the approach for installation of robotic operation system in cell manufacturing.

Understanding the formation and behaviors of droplets toward consideration of changeover during cell manufacturing.

Preventing the contamination of processed cells is required for achieving reproducible manufacturing. A droplet is one of the potential causes contamination in cell manufacturing. The present study elucidates the formation mechanism and characteristics of droplets based on the observation and detection of droplets on the base surface of the biological safety cabinet (BSC) where cell processing is conducted under unidirectional airflow. Pouring fluorescent solution into the vessel using a measuring pipette was conducted to visualize the formation of droplets by videos as well as visual detection by blacklight irradiation on the base surface of the BSC. The experiments revealed that airborne and non-airborne droplets emerged from bursting bubbles, which formed when the entire solution was pushed out of the measuring pipette. Therefore, the improving procedure of pouring technique when entire solution was not pushed out of the pipette realized no formation of the droplets due to the prevention of emergence of bubble. In addition, an alternative procedure in which the entire solution was poured into the deep point of the test tube prevented the flying of non-airborne droplets outside the tube, while airborne droplets that escaped the tube rode the airflow of BSC. These results suggested a method for the prevention of the droplet formation, as well as the deposit control of droplets onto the surface in BSC, leading to cleanup area in the BSC for changeover with environment continuity.

Variation in the manufacturing reproducibility of autologous cell-based products depending on raw material shipment conditions.

To prepare an autologous cell-based product in a cell processing facility, the raw material, which is collected from a patient, must first be shipped from a medical institution to the facility. The quality of this raw material varies depending on the patient, and variations due to transport methods also occur. Because the quality must be uniform and manufacturing processes need to be adjusted to account for these variations, determining the effect of shipment conditions on raw materials is very important for estimating cell manufacturability in the process design. In this study, a group of medical institutions located in different areas requested similar cell-based products processed by the same manufacturing method to a company that is licensed under the Act on the Safety of Regenerative Medicine in Japan. Manufacturing reproducibility was analyzed based on 456 cell batches received from two clinics that were processed used the same manufacturing method. The specific growth rates that were observed in the early growth phase supposed that the proliferative potential of the primary cells in the raw material was influenced by transit time. Simultaneously, the variation of the specific growth rates in the late phase were supposed to be hardly occurred. Thus, this study evaluated shipping conditions of the raw materials for an autologous cell-based product, and a strategy for verifying the influence of transportation on quality in manufacturing was suggested.

A novel, flexible and automated manufacturing facility for cell-based health care products: Tissue Factory.

INTRODUCTION: Current production facilities for Cell-Based Health care Products (CBHPs), also referred as Advanced-Therapy Medicinal Products or Regenerative Medicine Products, are still dependent on manual work performed by skilled workers. A more robust, safer and efficient manufacturing system will be necessary to meet the expected expansion of this industrial field in the future. Thus, the 'flexible Modular Platform (fMP)' was newly designed to be a true "factory" utilizing the state-of-the-art technology to replace conventional "laboratory-like" manufacturing methods. Then, we built the Tissue Factory as the first actual entity of the fMP. METHODS: The Tissue Factory was designed based on the fMP in which several automated modules are combined to perform various culture processes. Each module has a biologically sealed chamber that can be decontaminated by hydrogen peroxide. The asepticity of the processing environment was tested according to a pharmaceutical sterility method. Then, three procedures, production of multi-layered skeletal myoblast sheets, expansion of human articular chondrocytes and passage culture of human induced pluripotent stem cells, were conducted by the system to confirm its ability to manufacture CHBPs. RESULTS: Falling or adhered microorganisms were not detected either just after decontamination or during the cell culture processes. In cell culture tests, multi-layered skeletal myoblast sheets were successfully manufactured using the method optimized for automatic processing. In addition, human articular chondrocytes and human induced-pluripotent stem cells could be propagated through three passages by the system at a yield comparable to manual operations. CONCLUSIONS: The Tissue Factory, based on the fMP, successfully reproduced three tentative manufacturing processes of CBHPs without any microbial contamination. The platform will improve the manufacturability in terms of lower production cost, improved quality variance and reduced contamination risks. Moreover, its flexibility has the potential to adapt to the modern challenges in the business environment including employment issues, low operational rates, and relocation of facilities. The fMP is expected to become the standard design basis of future manufacturing facilities for CBHPs.

Effects of residual H2O2 on the growth of MSCs after decontamination.

INTRODUCTION: Regenerative therapy is a developing field in medicine. In the production of cell products for these therapies, hygienic management is even more critical than in the production of a chemical drug. At the same time, however, care is required with the use of decontamination agents, considering their effects on cell viability and characteristics. To date, hydrogen peroxide (H2O2) is most widely used for decontamination in pharmaceutical plants and cell processing facilities. METHODS: In this study, we examined the effects of residual H2O2 in the atmosphere of cell processing units after decontamination on the viability and proliferation of mesenchymal stem cells derived from human bone marrow. RESULTS: We detected residual H2O2 sufficient to affect cell proliferation and survival even more than 30 h after decontamination ended. Our results suggest a longer time period is required before starting operations after decontamination and that the operating time should be as short as possible. CONCLUSIONS: Here we show the effects of post-decontamination residual H2O2 on the viability and proliferation of mesenchymal stem cells derived from human bone marrow, which may provide us with important information about the hygienic management of cell processing facilities.

Experience of contamination during autologous cell manufacturing in cell processing facility under the Japanese Medical Practitioners Act and the Medical Care Act.

Cell therapy and regenerative medicine technologies require strict cell manufacturing procedures to be defined and addressed. Maintenance of the aseptic environment is critical to preclude extrinsic contamination risks, similar to conventional pharmaceutical manufacturing. However, intrinsic contamination risks exist in all cell manufacturing processes owing to the use of cells as the raw materials that cannot be sterilized, thus giving rise to the primary and secondary risks of cell contamination and cross-contamination, respectively. Analysis of contamination risks was conducted on experienced batches (29,858 batches) for the production of immune cells derived from autologous blood mononuclear cells under the Medical Practitioners Act and the Medical Care Act in Japan. From these batches, 0.06% (18 cases) of contamination occurred, representing low probability of contamination incidence during cell processing. Almost all the causes of these contaminations were regarded to be from the collected blood (intrinsic contamination), and subsequent cross-contaminations were prevented, considering that the secondary contamination risk can be reduced by adequate managements of operational procedures for changeover in aseptic environment.

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

Fabrication of human oral mucosal epithelial cell sheets for treatment of esophageal ulceration by endoscopic submucosal dissection.

BACKGROUND: Esophageal stenosis is one of the major complications of aggressive endoscopic resection. Tissue-engineered epithelial cell grafts have demonstrated effectiveness in promoting re-epithelialization and suppressing inflammation causing esophageal scarring and stenosis after endoscopic submucosal dissection (ESD) in an animal model. OBJECTIVE: To confirm the reproducibility and efficacy of a human oral mucosal epithelial cell (hOMEC) sheet cultured on temperature-responsive surface in conformity with Good Manufacturing Practice guidelines. DESIGN: A preclinical study. SETTING: Good Manufacturing Practice grade cell-processing center, animal laboratory. SUBJECTS: Canine esophageal ulcer models, which were made by ESD. INTERVENTIONS: Oral mucosal specimens were obtained from 7 healthy volunteers. MAIN OUTCOME MEASUREMENT: Fabricated and transplanted hOMEC sheets were subjected to histological analysis. RESULTS: The reproducibility of the fabrication of hOMEC sheets was confirmed. In this method, animal-derived materials such as 3T3 feeder layer and fetal bovine serum were successfully excluded from the culture condition. Furthermore, the environment of the culture room and safety cabinet in the cell-processing center was maintained for obtaining sterility assurances during the fabrication. Transplanted hOMEC sheets after ESD were observed to graft onto canine esophageal ulcer surfaces. LIMITATIONS: Small number of subjects, animal model. CONCLUSIONS: Cultured hOMEC sheets were fabricated without animal-derived materials and demonstrated efficacy as a medical device that promotes re-epithelialization of an esophageal ulcer after ESD.

Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study.

Periodontal-ligament-derived cells (PDL cells) have stem-cell-like properties and, when implanted into periodontal defects in vivo, can induce periodontal regeneration including the formation of new bone, cementum, and periodontal ligament. We have previously demonstrated that PDL cell sheets, harvested from temperature-responsive cell culture dishes, have a great potential for periodontal regeneration. The purpose of this study has been to validate the safety and efficacy of human PDL (hPDL) cell sheets for use in clinical trials. hPDL tissues from three donors were enzymatically digested, and the obtained cells were cultured with media containing autologous serum in a cell-processing center (CPC). The safety and efficacy of hPDL cell sheets were evaluated both in vitro and in vivo. In vitro studies showed that the hPDL cell sheets had high alkaline phosphatase activity and periostin expression (known PDL markers) and no contamination with microorganisms. In vivo studies revealed that hPDL cell sheets, implanted with dentin blocks, induced the formation of cementum and PDL-like tissue in immunodeficient mice. The hPDL cells presented no evidence of malignant transformation. Thus, hPDL cell sheets created in CPCs are safe products and possess the potential to regenerate periodontal tissues.

Liquid acrylate-endcapped biodegradable poly(epsilon-caprolactone-co-trimethylene carbonate). II. Computer-aided stereolithographic microarchitectural surface photoconstructs.

Advanced micromedical devices may require computer-aided photofabrication, by which microarchitectural surface design and entire macroshaped body design are feasible. Liquid acrylate-endcapped poly(epsilon-caprolactone-co-trimethylene carbonate)s, poly(CL/TMC)s, prepared using trimethylene glycol (TMG) or poly(ethylene glycol) (PEG) as an initiator and an acrylate group for subsequent terminal capping, were used as photocurable copolymers. The stereolithographically microarchitectured photoconstructs were prepared using a custom-designed apparatus with a moving ultraviolet (UV) light pen driven by a computer-assisted design program. The prepared photoconstructs included microneedles, a microcylinder and microbanks on surfaces. In vitro hydrolytic degradation proceeded with surface erosion when hydrophobic TMG-based photocured copolymers were employed, whereas very fast degradation of hydrophilic PEG-based photocured copolymers probably via concerted actions of surface erosion and bulk degradation was observed. In vivo hydrolytic behavior upon subcutaneous implantation in rats indicated that surface erosion proceeded for TMG-based photoconstructs. Anti-inflammatory drug (indomethacin) loading into microneedle-structured surfaces minimized inflammatory reactions. The possible biomedical microarchitectural three dimensinal in biomedical application photoconstructs was discussed.

Liquid acrylate-endcapped biodegradable poly(epsilon-caprolactone-co-trimethylene carbonate). I. Preparation and visible light-induced photocuring characteristics.

Photocurable liquid biodegradable copolymers were prepared by ring-opening copolymerization of epsilon-caprolactone (CL) and trimethylene carbonate (TMC) in the presence of a multifunctional hydroxyl group-bearing substance (di-, tri-, and tetra-functional alcohol and poly(ethylene glycol) (PEG) and its four-branched derivative) as an initiator and subsequent endcapping with acryloyl chloride at their hydroxyl terminals. These multifunctional, viscous liquid copolymers (molecular weights; approximately 2 x 10(3) to 7 x 10(3) g/mol) were converted to crosslinked solids by visible-light irradiation in the presence of camphorquinone as an initiator. The photocuring rate of these copolymers was enhanced by both higher functionality and lower molecule weight of the copolymers used. The photocuring rate depended on the amount of reducing agent (methacrylic acid 2-dimethylaminoethyl ester). Upon immersion in a phosphate buffer solution (pH 7.4), hydrolysis occurred preferentially on the surface except for photocured PEG-based copolymers that were degraded faster via both surface erosion and bulk degradation than low molecular weight alcohols-based copolymers. Cylindrical photocured constructs prepared by photoirradiation to the whole body in a mold filled with the liquid copolymer was demonstrated as an example of shape fabrication of biodegradable biomedical devices.

Liquid, phenylazide-end-capped copolymers of epsilon-caprolactone and trimethylene carbonate: preparation, photocuring characteristics, and surface layering.

Photoreactive phenylazide-end-capped liquid copolymers were prepared by ring-opening copolymerization of epsilon-caprolactone (CL) and trimethylene carbonate (TMC) at an equimolar monomer feed ratio in the presence of a polyol, namely, a low-molecular-weight alcohol (di-, tri-, and tetraol) or poly(ethylene glycol) (PEG) as an initiator and tin(II) 2-ethylhexanoate as a catalyst, followed subsequently by phenylazide derivatization at their hydroxyl terminus. These tri- and tetrabranched liquid copolymers (precursors) with a molecular weight from approximately 2500 to 7000 g/mol were cross-linked to yield insoluble solids by ultraviolet (UV) light irradiation. The photocuring rate increased with increasing functionality of phenylazide and UV intensity and decreasing thickness of the liquid film of precursors. The photo-cross-linkability of phenylazide-derivatized liquid copolymers was found to be higher than that of the corresponding coumarin-derivatized liquid copolymers. Poly(lactide) (PLA) films surface-layered with photocured copolymers were prepared by coating surfaces with phenylazide-derivatized copolymers and their subsequent photoirradiation. Endothelial cells adhered well on the nontreated PLA and low-molecular-weight alcohol-based copolymer-layered and photocured films. Little cell adhesion was observed on the hydrolytically surface-eroded PLA film and the PEG-based copolymer-layered film. When a phenylazide-derivatized hexapeptide with the cell-adhesion tripeptidyl sequence, Arg-Gly-Asp (RGD), common to cell adhesive proteins, was photoimmobilized on these surfaces, the surfaces became cell adhesive. Microarchitectured surfaces, which were prepared by sequential procedures of surface coating and photocuring using a photomask with lattice windows, produced regionally differentiated cell adhesiveness.

Synthesis and characterization of new chiral calix[4]arenes.

The unique chiral calixarenes were successfully synthesized by the following two methods: the Williamson ether synthesis and a stepwise ether cleavage with a mild Lewis acid. The high regioselectivity is recognized by the latter stepwise method. The ionophores having oligoethylene glycol unit efficiently extracted larger alkali metal ions like K+, Rb+, and Cs+ than smaller ones like Li+ and Na+. Their ion selectivity apparently changed by the chain length of crown ether. All racemates obtained could be resolved to each enantiomer by HPLC using a chiral column. The calixarenes with planar chirality recognized the chirality of guest molecules. Thus, the (-)-receptor resolved strongly forms 1:1 complex with (R)-(+)-alpha-phenylethylammonium picrate.

Liquid photocurable biodegradable copolymers: in vivo degradation of photocured poly(epsilon-caprolactone-co-trimethylene carbonate).

Liquid photoreactive poly(epsilon-caprolactone-co-trimethylene carbonate)s endcapped with a coumarin group [coumarinated poly(CL/TMC)s] were prepared using tetra-functional hydroxylated substances such as pentaerythritol or four-branched poly(ethylene glycol), b-PEG. These coumarinated copolymers are tetra-branched and exist as a viscous liquid (MW 5 x10(3) approximately 7 x 10(3)). They were photocured by ultraviolet (UV) light irradiation to obtain a swelling or nonswelling solid under water, depending on the type of initiator used. The resultant films were implanted into the subcutaneous tissues of rats for up to 5 months. The photocured b-PEG-based copolymer was completely degraded and sorbed within a 1 month. On the other hand, surface-eroding degradation of pentaerythritol-based, coumarinated poly(CL/TMC) progressed with implantation time, and minimal recruitment of neutrophils, macrophages, and multinucleated giant cells was observed over the implantation period. Among the pentaerythritol-based copolymers, the fastest surface erosion was observed for the copolymer with the highest epsilon-caprolactone content. Microfabricated films with microarrays in which photoconstructs were stereolithographically prepared, using three different coumarinated copolymers at different regions, showed that upon implantation there was regionally differentiated biodegradation of microarrays, and the degree of region-specific biodegradation depended on the type of photocured copolymer. The observed tendency for biodegradation was in good agreement with that observed during implantation of individual films in vivo. This study also demonstrates that the use of multi-material-arrayed films enables the determination of different responses in vivo using only one sample.

Photocurable liquid biodegradable copolymers: in vitro hydrolytic degradation behaviors of photocured films of coumarin-endcapped poly(epsilon-caprolactone-co-trimethylene carbonate).

Coumarin-endcapped tetrabranched liquid copolymers composed of epsilon-caprolactone and trimethylene carbonate (TMC), prepared using pentaerythritol or four-branched poly(ethylene glycol) (PEG) as an initiator, were ultraviolet irradiated to produce photocured solid biodegradable copolymers. The hydrolytic degradation behaviors of photocured films were determined from the weight loss of the films. The initial hydrolysis rate (determined for up to 24 h using a quartz crystal microbalance) was enhanced with aqueous solutions of higher pH. The hydrolysis rate in the early period of immersion was increased with an increase in TMC content, whereas that in the later period (week order) decreased with a increase in TMC content. This inverse relation of composition dependence on the hydrolysis rate between the early and late periods was discussed. Topological measurements using scanning electron microscopy and atomic force microscopy as well as depth profiles of the fluorescein-stained hydrolyzed layer showed that for the pentaerythritol-initiated copolymer, irrespective of copolymer composition, hydrolysis occurred at surface regions and surface erosion proceeded with immersion time. For PEG-based copolymers, both surface erosion and bulk degradation occurred simultaneously. The hydrolyzed surfaces became highly wettable with water and exhibited noncell adhesivity.