The Optimal Conditions of Chondrocyte Isolation and Its Seeding in the Preparation for Cartilage Tissue Engineering

Yonenaga, Kazumichi; Nishizawa, Satoru; Fujihara, Yuko; Asawa, Yukiyo; Kanazawa, Susumu; Nagata, Satoru; Takato, Tsuyoshi; Hoshi, Kazuto

doi:10.1089/ten.tec.2009.0597

Cited by 27 publications

(31 citation statements)

References 39 publications

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“…This may have been caused by some collagenase remaining in the medium of the primary culture when the washing step performed after enzymatic digestion was not sufficient. High concentrations and long incubation periods with collagenase are cytotoxic, as shown in a previous study (34). Another explanation for our observation is that a shortage of nutrition or growth factors may have occurred because of the decrease in the number of medium changes, which may have reduced the cell viability and adhesion of the chondrocytes.…”

Section: Discussionsupporting

confidence: 70%

“…At least 1 × 10 8 cells will be necessary for the production of regenerated cartilage of this size. Based on the results of a previous report regarding the concentration of collagenase required for digestion and the cell density at seeding, we consistently collected 1 × 10 6 cells from approximately 0.1 g of biopsied cartilage (34). When we seeded these cells at a density of 3,000-10,000 cells/cm 2 , we obtained over 1 × 10 7 cells at the end of the primary culture and reached the goal of over 1 × 10 8 cells on the subsequent passage.…”

Section: Resultsmentioning

confidence: 99%

“…Shortening the culture period may benefit patients by reducing the therapy period, thereby improving quality of life and health (8). We attempted to improve the efficacy of collagenase digestion for chondrocyte isolation from cartilage and to harvest the maximum possible number of viable cells (34). With this method, we successfully obtained 1 × 10 7 viable cells/g from the tissue, which was approximately 10-fold higher than the value previously reported (1 × 10 6 cells/g) (15).…”

mentioning

confidence: 89%

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Application of floating cells for improved harvest in human chondrocyte culture

Yonenaga¹,

Nishizawa²,

Fujihara³

et al. 2012

Biomed. Res.

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Cell culture medium, which must be discarded during medium change, may contain many cells that do not attach to culture plates. In the present study, we focused on these floating cells and attempted to determine their usefulness for cartilage regeneration. We counted the number of floating cells discarded during medium change and compared the proliferation and differentiation between floating cells and their adherent counterparts. Chondrocyte monolayer culture at a density of 5 × 10 3 cells/cm 2 produced viable floating cells at a rate of 2.7-3.2 × 10 3 cells/cm 2 per primary culture. When only the floating cells from one dish were harvested and replated in another dish, the number of cells was 2. ). The floating and adherent cells showed similar proliferation and differentiation properties. The recovery of floating cells from the culture medium could provide an approximately 1.5-fold increase in cell number over conventional monolayer culture. Thus, the collection of floating cells may be regarded as a simple, easy, and reliable method to increase the cell harvest for chondrocytes.

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Section: Discussionsupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 89%

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Application of floating cells for improved harvest in human chondrocyte culture

Yonenaga¹,

Nishizawa²,

Fujihara³

et al. 2012

Biomed. Res.

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“…Various methods have been utilized in order to achieve maximum harvest yield with optimal cell viability while preserving the chondrocyte phenotype [11][12][13]. Majority of these are implemented on animal chondrocytes that are usually used as control cells in the experiments with engineered constructs.…”

Section: Cells As Biomimetic System Componentmentioning

confidence: 99%

Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering

Gadjanski

2018

Osteochondral Tissue Engineering

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In order to engineer biomimetic osteochondral (OC) construct, it is necessary to address both the cartilage and bone phase of the construct, as well as the interface between them, in effect mimicking the developmental processes when generating hierarchical scaffolds that show gradual changes of physical and mechanical properties, ideally complemented with the biochemical gradients. There are several components whose characteristics need to be taken into account in such biomimetic approach, including cells, scaffolds, bioreactors as well as various developmental processes such as mesenchymal condensation and vascularization, that need to be stimulated through the use of growth factors, mechanical stimulation, purinergic signaling, low oxygen conditioning, and immunomodulation. This chapter gives overview of these biomimetic OC system components, including the OC interface, as well as various methods of fabrication utilized in OC biomimetic tissue engineering (TE) of gradient scaffolds. Special attention is given to addressing the issue of achieving clinical size, anatomically shaped constructs. Besides such neotissue engineering for potential clinical use, other applications of biomimetic OC TE including formation of the OC tissues to be used as high-fidelity disease/ healing models and as in vitro models for drug toxicity/efficacy evaluation are covered. Highlights Biomimetic OC TE uses "smart" scaffolds able to locally regulate cell phenotypes and dual-flow bioreactors for two sets of conditions for cartilage/ bone Protocols for hierarchical OC grafts engineering should entail mesenchymal condensation for cartilage and vascular component for bone Immunomodulation, low oxygen tension, purinergic signaling, time dependence of stimuli application are important aspects to consider in biomimetic OC TE Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering Ivana GadjanskiAbstract In order to engineer biomimetic osteochondral (OC) construct, it is necessary to address both the cartilage and bone phase of the construct, as well as the interface between them, in effect mimicking the developmental processes when generating hierarchical scaffolds that show gradual changes of physical and mechanical properties, ideally complemented with the biochemical gradients. There are several components whose characteristics need to be taken into account in such biomimetic approach, including cells, scaffolds, bioreactors as well as various developmental processes such as mesenchymal condensation and vascularization, that need to be stimulated through the use of growth factors, mechanical stimulation, purinergic signaling, low oxygen conditioning, and immunomodulation. This chapter gives overview of these biomimetic OC system components, including the OC interface, as well as various methods of fabrication utilized in OC biomimetic tissue engineering (TE) of gradient scaffolds. Special attention is given to addressing the issue of achieving clinical size, anatomically shaped constructs. Besides such neotissue en...

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“…The cartilage was cut into 1-mm 3 pieces using a scalpel and incubated in 0.15% collagenase solution at 37˚C for 24 hours with shaking in a thermostat [10]. The lysate was filtered through a cell strainer with a pore size of 100 μm to remove residues, and then centrifuged at 500 × g for 5 minutes to isolate human chondrocytes.…”

Section: Isolation and Analysis Of Chondrocytesmentioning

confidence: 99%

Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

Mori¹,

Kanazawa²,

Watanabe³

et al. 2013

MSA

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The efficiency of substance exchange may be decreased when the thickness and volume of such a tissue-engineered cartilage that is composed of cultured cells and porous scaffold increase. Moreover, during the transport of this construct with complicated shapes, excessive and focal mechanical loading may cause deformation. The establishment of incubation and transport methods is necessary for the three-dimensional tissue-engineered cartilage. Therefore, we investigated the preparation of an agarose mold with a concavity similar to the shape of 3-dimensional tissue-engineered cartilage to prevent excessive and focal concentration of stress, while avoiding interference with substance exchange as much as possible. Firstly, we investigated the preparation at 1% -4% agarose concentrations. Since the mechanical strength was insufficient at 1%, 2% was regarded as appropriate. Using 2% agarose, we prepared a mold with a 5 × 5 × 5 mm concavity to accommodate tissue-engineered cartilage (5 × 5 × 5 mm mixture of 1.5 × 10 7 cells and collagen gel), and stored the regenerative cartilage in it for 2 and 24 hours. On comparison with storage in a plastic mold with the same shape in which substance exchanged from side and bottom was impossible, although no significant differences were noted in the number or viability of cells after 2 hours, these were markedly reduced in the plastic mold after 24 hours. It was confirmed that favorable cell numbers and viability were maintained by immediately retaining the regenerative cartilage in the culture medium in the agarose mold and keeping the temperature at 37˚C. Since this agarose mold also buffers against mechanical forces loaded on the three-dimensional regenerative tissue, it may be useful as a container for storage and transport of large-sized three-dimensional regenerative tissue.

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The Optimal Conditions of Chondrocyte Isolation and Its Seeding in the Preparation for Cartilage Tissue Engineering

Cited by 27 publications

References 39 publications

Application of floating cells for improved harvest in human chondrocyte culture

Application of floating cells for improved harvest in human chondrocyte culture

Mimetic Hierarchical Approaches for Osteochondral Tissue Engineering

Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

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