Application of floating cells for improved harvest in human chondrocyte culture

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

doi:10.2220/biomedres.33.281

Cited by 4 publications

(5 citation statements)

References 35 publications

(39 reference statements)

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“…In primary culture, the maximum number of cells obtained from 0.1 g of articular cartilage after 1 week (1.5 × 10 5 ) was less than that obtained from 0.1 g of auricular cartilage after the same culture duration (6.0 × 10 5 ) [3] . A previous study that compared chondrocytes from different cartilage tissues [17] , [25] , [26] , [27] , [28] , [29] , [30] found that the proliferative abilities of same-aged articular and auricular cartilages were different after 3 weeks of culture; however, no difference in the proliferative abilities of same-aged articular and auricular cartilages was observed after a 1-week culture period in one study [17] .…”

Section: Discussionmentioning

confidence: 81%

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Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues

Yonenaga¹,

Nishizawa²,

Nakagawa

et al. 2017

Regenerative Therapy

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IntroductionThere are various types of cartilage, including the auricular and articular cartilages. These cartilages have different functions, and their matrix volume and density of chondrocytes may differ. Thus, different protocols may be required to digest different types of cartilage.MethodsIn this study, we examined protocols for the digestion of articular and auricular cartilages and determined the optimal conditions for articular cartilage digestion.ResultsOur histological findings showed that the articular cartilage has a larger matrix area and fewer cells than the auricular cartilage. In 1-mm2 areas of articular and auricular cartilages, the average numbers of cells were 44 and 380, respectively, and the average matrix areas were 0.94 and 0.77 mm2, respectively. The maximum numbers of viable cells (approximately 1 × 105 cells/0.1 g of tissue) were obtained after digestion in 0.15, 0.3, or 0.6% collagenase for 24 h, in 1.2% collagenase for 6 h, or in 2.4% collagenase for 4 h. In tissues incubated in 0.15 or 0.3% collagenase, the cell numbers were lower than 1 × 105, even at 24 h, possibly reflecting incomplete digestion of cartilage. No significant differences were observed in the results of apoptosis assays for all collagenase exposure times and concentrations. However, cell damage appeared to be greater when collagenase concentrations were high. When cells obtained after digestion with different concentrations of collagenase were seeded at a density of 3000 cells/cm2, they yielded the maximum cell numbers after 1 week.ConclusionsWe recommend a 24-h incubation in 0.6% collagenase as the optimal condition for chondrocyte isolation from articular cartilage. Moreover, we found that the optimum cell-seeding density is approximately 3000 cells/cm2. Conditions determined in this study would maximize the yield of isolated articular chondrocytes and enable the generation of a large quantity of cultured cells.

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Section: Discussionmentioning

confidence: 81%

“…Collagenase digestion is the first step in cartilage tissue engineering [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] . Incomplete digestion reduces the efficiency of tissue engineering and affects the safety of the process and stability of grafts.…”

Section: Introductionmentioning

confidence: 99%

Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues

Yonenaga¹,

Nishizawa²,

Nakagawa

et al. 2017

Regenerative Therapy

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“…The growth of cells in aggregates was examined under normal cell culture conditions. Floating cells need to be slowly agitated while culturing to ensure their oxygen supply. − In addition, when collecting the proliferated cells, it is necessary to carefully centrifuge them, and processing them in large amounts is difficult. However, in our procedure, if the cells are aggregated and cultured using magnetic particles and then recovered using the magnetic field, such as medium exchange, the operation becomes simpler and more automatic, even with floating cells.…”

Section: Results and Discussionmentioning

confidence: 99%

“…In traditional cell culture procedures, for preparing cell suspensions, floating cells are suspended in a cell culture medium and cultured with slow stirring. − On the other hand, adherent cells need to be treated with enzyme after culturing and proliferating on a culture plate. , It is to decompose the proteins contributed to cell adhesion and the secreted extracellular matrix . After detachment of the cells, a centrifugation process should be needed.…”

Section: Introductionmentioning

confidence: 99%

Preparation of Magnetic Hydrogel Microparticles with Cationic Surfaces and Their Cell-Assembling Performance

Ishihara

Narita

Teramura

et al. 2021

ACS Biomater. Sci. Eng.

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Cationic magnetic hydrogel microparticles with high retention on cell surfaces were prepared using a two-step procedure. Using these magnetic hydrogel microparticles, cells were clustered with each other, and cell aggregates were prepared effectively. Cross-linked poly(vinyl alcohol) (PVA) hydrogel microparticles containing iron oxide nanoparticles were prepared. The diameter of the microparticles was in the range of 200–500 nm. Water-soluble cationic polymers containing both trimethyl ammonium (TMA) groups and phenylboronic acid (PBA) groups were synthesized for the surface modification of the microparticles. To regulate the composition, electrically neutral phosphorylcholine groups were introduced into the polymer. Covalent bonds were formed between the hydroxy groups of PVA microparticles and PBA groups in the polymer. The surface zeta potential of the microparticles reflected the composition of the TMA groups. The particles responded to an external magnetic field and clustered rapidly. Microparticles were adsorbed on the floating cell surface and induced cell aggregation quickly when a magnetic field was applied. Under the most effective conditions, the diameter of the cell aggregates increased to approximately 1 mm after 30 min. Denser cell aggregates were formed by the synergistic effects of the magnetic field and the properties of the microparticles. The formed cell aggregates continued to grow for more than 4 days under an applied magnetic field, indicating that the ability of the cells in the aggregate to proliferate was well maintained.

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“…A variety of techniques have been implemented to minimize excessive cell loss during the cell extraction and expansion procedures, including the re-inoculation of floating cells in monolayer culture [11] , and the addition of enzymes to the cell pellet to increase the effectiveness of resuspension after centrifugation [12] . However, the addition of enzymes necessitates an accruement of an additional centrifugation step within the procedure, in effect, causing potential negative impacts on cell survival.…”

Section: Introductionmentioning

confidence: 99%

Improving chondrocyte harvests with poly(2-hydroxyethyl methacrylate) coated materials in the preparation for cartilage tissue engineering

Harata

Watanabe

Nagata³

et al. 2017

Regenerative Therapy

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Remarkable advances have been made in cartilage regenerative medicine to cure congenital anomalies including microtia, tissue defects caused by craniofacial injuries, and geriatric diseases such as osteoarthritis. However, those procedures require a substantial quantity of chondrocytes for tissue engineering. Previous studies have required several passages to obtain sufficient cell numbers for three-dimensional and monolayer cultures. Thus, our objective was to improve the quantity of chondrocytes that can be obtained by examining an anti-fouling polyhydrophilic chemical called poly(2-hydroxyethyl methacrylate) (pHEMA). To determine the effectiveness of the chemical, pHEMA solution was applied via dip-coating to centrifuge tubes, serological pipettes, and pipette tips. The cell quantity obtained during standard cell culturing and passaging procedures was measured alongside non-coated materials as a control. A significant 2.2-fold increase of chondrocyte yield was observed after 2 passages when pHEMA was applied to the tubes compared to when non-coated tubes were utilized. The 3-dimensional chondrocyte pellets prepared from the respective cell populations and transplanted into nude mice were histologically and biochemically analyzed. No evidence of difference in matrix production for in vitro and in vivo cultures was found as well as similar proliferation rates and colony formation abilities. The use of pHEMA provides a powerful alternative method for expanding the quantity of chondrocytes harvested and handled during cell isolation and passaging to enhance cartilage tissue engineering.

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Application of floating cells for improved harvest in human chondrocyte culture

Cited by 4 publications

References 35 publications

Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues

Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues

Preparation of Magnetic Hydrogel Microparticles with Cationic Surfaces and Their Cell-Assembling Performance

Improving chondrocyte harvests with poly(2-hydroxyethyl methacrylate) coated materials in the preparation for cartilage tissue engineering

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