The properties of bioengineered chondrocyte sheets for cartilage regeneration

<p>Abstract</p> <p>Background</p> <p>Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage.</p> <p>Results</p> <p>The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets.</p> <p>Conclusion</p> <p>The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.</p

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. Keywords: Regenerative medicine, Automation, Cell processing facility, Manufacturing, Decontaminatio

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

<div><p>Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.</p></div

Cardiac differentiation of ES cells in the bioreactor.

<p>(A) Real-time PCR analysis (<i>n</i> = 3). Solid lines indicate the results of continuous medium exchange, and dotted lines indicate the results of intermittent medium exchange. *<i>p</i><0.05. (B) Representative dot plots of flow cytometric analyses of GFP(+) cells in EBs at day 10. The graph shows the percentage of GFP(+) cells at day 10 (<i>n</i> = 6). (C) GFP(+) cells were collected using a fluorescence-activated cell sorter and then seeded onto gelatin-coated dishes. GFP(+) cells expressed sarcomeric α-actinin (upper), myosin heavy chain (MHC, middle) and cardiac troponin T (lower) in a fine striated pattern. Nuclei were counterstained with DAPI. Scale bars, 20 µm. (D, E) Comparison of the numbers of cardiomyocytes among culture conditions using R1 ES cells. (D) The number of remaining cells 8 days after starting culture with G418 (<i>n</i> = 3). Data are means ± s.d. (E) At 8 days after starting culture with G418 in the bioreactor with or without regulation, the cells were dissociated and seeded onto 1% gelatin-coated 24-well plates. At 24 h after seeding, the cells were fixed and immunostained for sarcomeric-α actinin. The percentage of sarcomeric α-actinin-positive cells among the remaining cells was calculated and is shown in the graph (<i>n</i> = 3). Data are means ± s.d. Right, representative images. Nuclei were stained with DAPI. Bars, 200 µm. (F) Comparison of cardiomyocyte engraftment between medium exchange systems. Purified cardiomyocytes after treatment with G418 were co-cultured with fibroblasts at the ratio of 8∶2. At day 2, cells were fixed and immunostained with cTnT. The percentage of cTnT-positive cells was calculated and is shown in the graph (<i>n</i> = 3). Data are means ± s.d. n.s., not significant. Right, representative images. Nuclei were stained with Hoechst33258. Bars, 200 µm.</p