Adult human articular chondrocytes in a microcarrier-based culture system: expansion and redifferentiation (original) (raw)
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Can Microcarrier-Expanded Chondrocytes Synthesize Cartilaginous Tissue In Vitro?
Tissue Engineering Part a, 2011
Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on MillicellÔ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.
European cells & materials, 2012
Cell-based cartilage repair strategies such as matrix-induced autologous chondrocyte implantation (MACI) could be improved by enhancing cell performance. We hypothesised that micro-aggregates of chondrocytes generated in high-throughput prior to implantation in a defect could stimulate cartilaginous matrix deposition and remodelling. To address this issue, we designed a micro-mould to enable controlled high-throughput formation of micro-aggregates. Morphology, stability, gene expression profiles and chondrogenic potential of micro-aggregates of human and bovine chondrocytes were evaluated and compared to single-cells cultured in micro-wells and in 3D after encapsulation in Dextran-Tyramine (Dex-TA) hydrogels in vitro and in vivo. We successfully formed micro-aggregates of human and bovine chondrocytes with highly controlled size, stability and viability within 24 hours. Micro-aggregates of 100 cells presented a superior balance in Collagen type I and Collagen type II gene expression...
Journal of Tissue Engineering and Regenerative Medicine, 2009
Biodegradable macroporous gelatin microcarriers fixed with blood-derived biodegradable glue is proposed as a delivery system for human autologous chondrocytes. Cell-seeded microcarriers were embedded in four biological gluesrecalcified citrated whole blood, recalcified citrated plasma with or without platelets, and a commercially available fibrin glue -and cultured in an in vitro model under static conditions for 16 weeks. No differences could be verified between the commercial fibrin glue and the blood-derived alternatives. Five further experiments were conducted with recalcified citrated platelet rich plasma alone as microcarrier sealant, using two different in vitro culture models and chondrocytes from three additional donors. The microcarriers were found chondrocyte adhesion and expansion, as well as extracellular matrix synthesis. Matrix formation occurred predominantly at sample surfaces under the static conditions. The presence of microcarriers proved essential for the glues to support the structural takeover of extracellular matrix proteins produced by the embedded chondrocytes, as exclusion of the microcarriers resulted in unstable structures that dissolved before matrix formation could occur. Immunohistochemical analysis revealed presence of SOX-9 and S-100 positive chondrocytes as well as production of aggrecan and collagen type I, but not of the cartilage-specific collagen type II. These results imply that blood-derived glues are indeed potentially applicable for encapsulation of chondrocyte-seeded microcarriers. However, the static in vitro models used in this study proved incapable of supporting cartilage formation throughout the engineered constructs. Aigner T, Gebhard PM, Schmid E, et al. 2003; SOX9 expression does not correlate with type II collagen expression in adult articular chondrocytes. Matrix Biol 22(4): 363-372. Akeda K, An HS, Okuma M, et al. 2006; Platelet-rich plasma stimulates porcine articular chondrocyte proliferation and matrix biosynthesis. Osteoarthritis Cartilage 14(12): 1272-1280. Blunk T, Sieminski AL, Gooch KJ, et al. 2002; Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng 8(1): 73-84. Borg DJ, Dawson RA, Leavesley DI, et al. 2009; Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture. J Biomed Mater Res A 88(1): 184-194. Boyan BD, Hummert TW, Dean DD, et al. 1996; Role of material surfaces in regulating bone and cartilage cell response. Biomaterials 17(2): 137-146. Brittberg M, Sjögren-Jansson E, Lindahl A, et al. 1997; Influence of fibrin sealant (Tisseel) on osteochondral defect repair in the rabbit knee. Biomaterials 18(3): 235-242. Chun KW, Yoo HS, Yoon JJ, et al. 2004; Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function. Biotechnol Prog 20(6): 1797-1801. Chung HJ, Kim IK, Kim TG, et al. 2008; Highly open porous biodegradable microcarriers: in vitro cultivation of chondrocytes for injectable delivery. Tissue Eng Part A 14(5): 607-615. Demetriou AA, Reisner A, Sanchez J, et al. 1988; Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology 8(5): 1006-1009. Drengk A, Zapf A, Sturmer EK, et al. 2008; Influence of Platelet-Rich Plasma on Chondrogenic Differentiation and Proliferation of Chondrocytes and Mesenchymal Stem Cells. Cells Tissues Organs Eyrich D, Brandl F, Appel B, et al. 2007; Long-term stable fibrin gels for cartilage engineering. Biomaterials 28(1): 55-65. Fernandes AM, Fernandes TG, Diogo MM, et al. 2007; Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system. J Biotechnol 132(2): 227-236. Frondoza C, Sohrabi A, Hungerford D. 1996; Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomaterials 17(9): 879-888. Gaissmaier C, Fritz J, Krackhardt T, et al. 2005; Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures. Biomaterials 26(14): 1953-1960. Goessl A, Redl H. 2005; Optimized thrombin dilution protocol for a slowly setting fibrin sealant in surgery. Eur Surg 37(1): 43-51. Holtzer H, Abbott J, Lash J, et al. 1960; The Loss of Phenotypic Traits by
2012
Autologous Cell Implantation (ACI) is a currently practiced cell-based therapy to repair cartilage defects. Several strategies have been explored to expand the number of chondrocytes ex vivo. However, these methods are unable to provide sufficient quantity of chondrocytes with unaltered phenotype. To maintain the original phenotype in monolayer culture and to expand cell proliferation, primary human chondrocytes isolated by enzymatic digestion were cultured in a DMEM medium supplemented with Ac-Gly-Gly-OH dipeptide The aim of our study was to investigate and compare by flow cytometry the viability and the cell proliferation of chondrocytes obtained by culture in medium containing the dipeptide with the cells cultured in a classical system. The results we obtained provide that proliferation and viability of chondrocytes cultured in presence of DMEM medium containing Ac-Gly-Gly-OH were higher and thus can be used in the culture of chondrocytes devoted to reconstructive clinical proced...
Experimental biology and medicine (Maywood, N.J.), 2017
Personalized features in the treatment of knee injuries and articular replacement therapies play an important role in modern life with increasing demand. Therefore, cell-based therapeutic approaches for the regeneration of traumatic defects of cartilage tissue were developed. However, great variations in the quality of repair tissue or therapeutic outcome were observed. The aim of the study was to capture and visualize individual differentiation capacities of chondrocytes derived from different donors with regard to a possible personal regeneration capacity using a cell-based therapy. The redifferentiation potential of monolayer cultured cells was analyzed in a scaffold-free three-dimensional tissue model. Furthermore, stimulating options using cartilage maturation factors such as L-ascorbic acid and transforming growth factor beta 2 (TGF-β2) on this process were of special interest. Cells and tissues were analyzed via histological and immunohistochemical methods. Gene expression wa...
Cell Transplantation, 2008
The main purpose of this work has been to establish a new culturing technique to improve the chondrogenic commitment of isolated adult human chondrocytes, with the aim of being used during cell-based therapies or tissue engineering strategies. By using a rather novel technique to generate scaffold-free three-dimensional (3D) structures from in vitro expanded chondrocytes, we have explored the effects of different culture environments on cartilage formation. Three-dimensional chondrospheroids were developed by applying the hanging-drop technique. Cartilage tissue formation was attempted after combining critical factors such as serumcontaining or serum-free media and atmospheric (20%) or low (2.5%) oxygen tensions. The quality of the formed microtissues was analyzed by histology, immunohistochemistry, electron microscopy, and real-time PCR, and directly compared with native adult cartilage. Our results revealed highly organized, 3D tissue-like structures developed by the hanging-drop method. All culture conditions allowed formation of 3D spheroids; however, cartilage generated under low oxygen tension had a bigger size, enhanced matrix deposition, and higher quality of cartilage formation. Real-time PCR demonstrated enhanced expression of cartilage-specific genes such us collagen type II and aggrecan in 3D cultures when compared to monolayers. Cartilage-specific matrix proteins and genes expressed in hanging-drop-developed spheroids were comparable to the expression obtained by applying the pellet culture system. In summary, our results indicate that a combination of 3D cultures of chondrocytes in hanging drops and a low oxygen environment represent an easy and convenient way to generate cartilage-like microstructures. We also show that a new specially tailored serum-free medium is suitable for in vitro cartilage tissue formation. This new methodology opens up the possibility of using autogenously produced solid 3D structures with redifferentiated chondrocytes as an attractive alternative to the currently used autologous chondrocyte transplantation for cartilage repair.
Tissue Engineering Part A, 2009
Objective: To determine if selected culture conditions enhance the expansion and redifferentiation of chondrocytes isolated from human osteoarthritic cartilage with yields appropriate for creation of constructs for treatment of joint-scale cartilage defects, damage, or osteoarthritis. Methods: Chondrocytes isolated from osteoarthritic cartilage were analyzed to determine the effects of medium supplement on cell expansion in monolayer and then cell redifferentiation in alginate beads. Expansion was assessed as cell number estimated from DNA, growth rate, and day of maximal growth. Redifferentiation was evaluated quantitatively from proteoglycan and collagen type II content, and qualitatively by histology and immunohistochemistry. Results: Using either serum or a growth factor cocktail (TFP: transforming growth factor b1, fibroblast growth factor 2, and platelet-derived growth factor type bb), cell growth rate in monolayer was increased to 5.5Â that of corresponding conditions without TFP, and cell number increased 100-fold within 17 days. In subsequent alginate bead culture with human serum or transforming growth factor b1 and insulin-transferrin-selenium-linoleic acidbovine serum albumin, redifferentiation was enhanced with increased proteoglycan and collagen type II production. Effects of human serum were dose dependent, and 5% or higher induced formation of chondron-like structures with abundant proteoglycan-rich matrix. Conclusion: Chondrocytes from osteoarthritic cartilage can be stimulated to undergo 100-fold expansion and then redifferentiation, suggesting that they may be useful as a cell source for joint-scale cartilage tissue engineering.
Isolation of Chondrocytes from Human Cartilage and Cultures in Monolayer and 3D
Methods in molecular biology, 2020
Chondrocytes are the only cell type in cartilage. The dense cartilage extracellular matrix surrounding the chondrocytes makes isolating these cells a complex and lengthy task that subjects the cells to harsh conditions. Protocols to isolate expand and maintain these cells have been improved over the years, providing ways to obtain viable cells for tissue engineering and clinical applications. Here we describe a method to obtain populations of chondrocytes that are able to expand and maintain a native-like phenotype.