Expansion Of Human Articular Chondrocytes Research Articles

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

IntroductionCurrent 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.MethodsThe 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.ResultsFalling 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.ConclusionsThe 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.

Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.

Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-ss had an inhibitory effect on microcarrier expanded chondrocytes. Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair.

TGF β‐1 administration during Ex vivo expansion of human articular chondrocytes in a serum‐free medium redirects the cell phenotype toward hypertrophy

Cell-based cartilage resurfacing requires ex vivo expansion of autologous articular chondrocytes. Defined culture conditions minimize expansion-dependent phenotypic alterations but maintenance of the cells' differentiation potential must be carefully assessed. Transforming growth factor β-1 (TGF β-1) positively regulates the expression of several cartilage proteins, but its therapeutic application in damaged cartilage is controversial. Thus we evaluated the phenotypic outcomes of cultured human articular chondrocytes exposed to TGF β-1 during monolayer expansion in a serum-free medium. After five doublings cells were transferred to micromass cultures to assess their chondrogenic differentiation, or replated in osteogenic medium. Immunocytostainings of micromasses of TGF-expanded cells showed loss of aggrecan and type II collagen. Positivity was evidenced for RAGE, IHH, type X collagen and for apoptotic cells, paralleling a reduction of BCL-2 levels, suggesting hypertrophic differentiation. TGF β-1-exposed cells also evidenced increased mRNA levels for bone sialoprotein, osteopontin, matrix metalloproteinase-13, TIMP-3, VEGF and SMAD7, enhanced alkaline phosphatase activity and pyrophosphate availability. Conversely, SMAD3 mRNA and protein contents were reduced. After osteogenic induction, only TGF-expanded cells strongly mineralized and impaired p38 kinase activity, a contributor of chondrocytes' differentiation. To evaluate possible endochondral ossification progression, we seeded the chondrocytes on hydroxyapatite scaffolds, subsequently implanted in an in vivo ectopic setting, but cells failed to reach overt ossification; nonetheless, constructs seeded with TGF-exposed cells displayed blood vessels of the host vascular supply with enlarged diameters, suggestive of vascular remodeling, as in bone growth. Thus TGF-exposure during articular chondrocytes expansion induces a phenotype switch to hypertrophy, an undesirable effect for cells possibly intended for tissue-engineered cartilage repair.

Cartilage-Characteristic Matrix Reconstruction by Sequential Addition of Soluble Factors During Expansion of Human Articular Chondrocytes and Their Cultivation in Collagen Sponges

Articular cartilage has a poor capacity for spontaneous repair. Tissue engineering approaches using biomaterials and chondrocytes offer hope for treatments. Our goal was to test whether collagen sponges could be used as scaffolds for reconstruction of cartilage with human articular chondrocytes. We investigated the effects on the nature and abundance of cartilage matrix produced of sequential addition of chosen soluble factors during cell amplification on plastic and cultivation in collagen scaffolds. Isolated human articular chondrocytes were amplified for two passages with or without a cocktail of fibroblast growth factor (FGF)-2 and insulin (FI). The cells were then cultured in collagen sponges with or without a cocktail of bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine (BIT). The constructs were cultivated for 36 days in vitro or for another 6-week period in a nude mouse-based contained-defect organ culture model. Gene expression was analyzed using polymerase chain reaction, and protein production was analyzed using Western-blotting and immunohistochemistry. Dedifferentiation of chondrocytes occurred during cell expansion on plastic, and FI stimulated this dedifferentiation. We found that addition of BIT could trigger chondrocyte redifferentiation and cartilage-characteristic matrix production in the collagen sponges. The presence of FI during cell expansion increased the chondrocyte responsiveness to BIT.

Cell expansion of human articular chondrocytes on macroporous gelatine scaffolds—impact of microcarrier selection on cell proliferation

This study investigates human chondrocyte expansion on four macroporous gelatine microcarriers (CultiSpher) differing with respect to two manufacturing processes—the amount of emulsifier used during initial preparation and the gelatine cross-linking medium. Monolayer-expanded articular chondrocytes from three donors were seeded onto the microcarriers and cultured in spinner flask systems for a total of 15 days. Samples were extracted every other day to monitor cell viability and establish cell counts, which were analysed using analysis of variance and piecewise linear regression. Chondrocyte densities increased according to a linear pattern for all microcarriers, indicating an ongoing, though limited, cell proliferation. A strong chondrocyte donor effect was seen during the initial expansion phase. The final cell yield differed significantly between the microcarriers and our results indicate that manufacturing differences affected chondrocyte densities at this point. Remaining cells stained positive for chondrogenic markers SOX-9 and S-100 but extracellular matrix formation was modest to undetectable. In conclusion, the four gelatine microcarriers supported chondrocyte adhesion and proliferation over a two week period. The best yield was observed for microcarriers produced with low emulsifier content and cross-linked in water and acetone. These results add to the identification of optimal biomaterial parameters for specific cellular processes and populations.

Controlling Medium Osmolality Improves the Expansion of Human Articular Chondrocytes in Serum-Free Media

To investigate the effects of medium osmolality on the expansion of human articular chondrocytes (HACs) with serum-free media (SFM), proprietary SFM of various osmolalities (290, 320, 350, 400, and 450 mOsm/kg), supplemented with components known to enhance chondrocyte growth, were constructed by the adjustment of NaCl concentration. It was found that HACs obtained better expansion in SFM at osmolalites lower than the average osmolality (400 mOsm/kg) of human articular cartilage in vivo. SFM at 290, 320, and 350 mOsm/kg showed similar growth, attaining up to a 1.55-fold increase in the proliferation rate compared with SFM at 400 mOsm/kg. Increasing SFM osmolality to 450 mOsm/kg resulted in a proliferation rate of 0.65-fold lower than at 400 mOsm/kg. Chondrogenic capacity was also examined via three-dimensional pellet cultures in a chondrogenic medium with HACs expanded in 320 and 400 mOsm/kg SFM. Biochemical, histological, and immunohistochemical analyses revealed similar glycosaminoglycan and collagen type II contents in both groups. Taken together, these results show that the expansion of HACs in SF cultures can be improved by adjusting the medium osmolality to be within the range of 290-350 mOsm/kg and that controlling medium osmolality during monolayer cultures does not deter the tissue-forming capability of the cells.

Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: A step towards exploring a potential use of matrix-induced cell therapy

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and ‘chondrocytes-fibrin’ construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM + 10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM + 2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM + 10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs > FD2GFs > FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and ‘chondrocytes-fibrin’ constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, ‘chondrocytes-fibrin’ constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality ‘chondrocytes-fibrin’ constructs which may have potential use of matrix-induced cell implantation.

Differential Protein Expression in Human Articular Chondrocytes Expanded in Serum-Free Media of Different Medium Osmolalities by DIGE

Expansion of human articular chondrocytes (HACs) in serum-free medium (SFM) has been shown to be enhanced by the control of medium osmolality. HACs attained better growth when cultured at osmolalities lower than the average osmolality found in articular cartilage in vivo, although the specific causes for this improved expansion were not speculated. The aim of this study was to perform monolayer cultures of HACs in SFM at two different medium osmolalities, 320 and 400 mOsm/kg, and observe changes in protein content. A proteomics approach using differential in gel electrophoresis (DIGE) revealed differences in 20 spots. MALDI-TOF/TOF mass spectrometry identified 18 of the 20 spots. Proteins affected by higher osmolality in SFM were identified and found to function, traditionally, in the cytoskeleton, protection against reactive oxygen species, mRNA biogenesis, and the heat shock response. All of these proteins were correlated with the cell cycle and proliferation, emphasizing the importance of medium osmolality control in the expansion of HACs in SFM. In addition, no protein expression changes were observed regarding chondrogenic markers, which supports our previous result that adjusting medium osmolality in SFM, while enhancing growth, does not deter the tissue-forming capability of HACs.

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

Evaluation of Cartilage Specific Matrix Synthesis of Human Articular Chondrocytes after Extended Propagation on Microcarriers by Image Analysis

Background Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. Methods Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. Results The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-β had an inhibitory effect on microcarrier expanded chondrocytes. Conclusions Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

Autologous versus Pooled Human Serum for Articular Chondrocyte Growth

To evaluate the effect of autologous human serum (AHS) versus pooled human serum (PHS) versus foetal bovine serum (FBS) for growth of articular chondrocytes and formation of chondrocytefibrin constructs. Experiments with monolayer culture expansion of human articular chondrocytes were performed using basic culture media supplemented with 10% AHS, PHS, or FBS. Growth kinetics and specific phenotypic expression of the serially expanded chondrocytes were evaluated. Large-scale culture expansion was used to obtain about 30 million cells to form chondrocyte-fibrin constructs. All constructs were implanted subcutaneously at the dorsum part of athymic nude mice for 8 weeks. The in vivo constructs were evaluated using histological and gene expression studies. The morphology of primary cultured chondrocytes (P0) was polygonal and became more elongated and larger after serial passages (P1, P2, and P3). This was comparable for AHS, PHS, and FBS. Total cell yields accumulated for AHS (28 million) and PHS (41 million) were significantly higher than those for FBS (4 million). After 8 weeks of implantation, in vivo chondrocyte-fibrin constructs demonstrated a glistening white and firm texture, comparable to normal hyaline cartilage. All constructs exhibited histo-architectural characteristics of well-distributed cartilage-isolated cells embedded within basophilic ground substance. Presence of accumulated proteoglycans cartilage-rich matrix was indicated by positive orange-red Safranin O staining. During monolayer culture expansion, collagen type II gene expression was down-regulated, while collagen type I gene expression was up-regulated. Collagen type II--the specific chondrogenesis marker--was re-expressed in the in vivo chondrocyte-fibrin construct. AHS and PHS are better than FBS for in vitro cultivation of human articular chondrocytes.

Secretion of matrix metalloproteinase 3 by expanded articular chondrocytes as a predictor of ectopic cartilage formation capacity in vivo

Monolayer expansion of human articular chondrocytes (HACs) is known to result in progressive dedifferentiation of the chondrocytes and loss of their stable cartilage formation capacity in vivo. For an optimal outcome of chondrocyte-based repair strategies, HACs capable of ectopic cartilage formation may be required. This study was undertaken to identify secreted candidate molecules, in supernatants of cultured HACs, that could serve as predictors of the ectopic cartilage formation capacity of cells. Standardized medium supernatants (n = 5 knee cartilage samples) of freshly isolated HACs (PD0) and of HACs expanded for 2 or 6 population doublings (PD2 and PD6, respectively) were screened by a multiplexed immunoassay for 15 distinct interleukins, 8 matrix metalloproteinases (MMPs), and 11 miscellaneous soluble factors. Cartilage differentiation markers such as cartilage oligomeric matrix protein and YKL-40 were determined by enzyme-linked immunosorbent assay. HACs from each culture were subcutaneously transplanted into SCID mice, and the capacity of the chondrocytes to form stable cartilage was examined histologically 4 weeks later. Whereas freshly isolated (PD0) HACs generated stable ectopic cartilage that was positive for type II collagen, none of the cell transplants at PD6 formed cartilaginous matrix. Loss of the ectopic cartilage formation capacity between PD0 and PD6 correlated with a drop in the secretion of MMP-3 to <10% of initial levels, whereas changes in the other investigated molecules were not predictive. Chondrocytes with MMP-3 levels of >or=20% of initial levels synthesized cartilaginous matrix, whereas those with low MMP-3 levels (<10% of initial levels) at PD2 failed to regenerate ectopic cartilage. Loss of the capacity for stable ectopic cartilage formation in the course of HAC dedifferentiation can be predicted by determining the relative levels of MMP-3, demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.

Growth Factors for Clinical-Scale Expansion of Human Articular Chondrocytes: Relevance for Automated Bioreactor Systems

The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 cells/cm2 in medium containing 10% fetal bovine serum or 10,000 cells/cm2 with 1%, 5%, or 10%HS. The chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems.

P175 Administration of TGF beta-1 during the expansion of human articular chondrocytes may trigger the ontogenesis of endochondral bone formation in the cultured cells

P175 Administration of TGF beta-1 during the expansion of human articular chondrocytes may trigger the ontogenesis of endochondral bone formation in the cultured cells