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Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Cells grown in monolayer will continue to proliferate until they cover the tissue culture dish and reach confluency. When cells reach confluency, or shortly beforehand, cells must be passaged or otherwise subcultured. Failure to do so will result in reduced proliferation or cell death. To do this, the cells must first be detached from the substrate using a proteolytic enzyme, commonly trypsin, although collagenase and dispase may also be used. This suspension of cells is then counted, fractioned, and used to reseed new tissue culture plates, resulting in a single passage. As discussed before, with increasing culture time and passage, chondrocytes dedifferentiate. It should also be noted that aside from stem cells and immortalized cell lines, most cells can only be passaged a set number of times before reaching senescence.
Microbiological Considerations
Published in Anne F. Booth, Sterilization Validation & Routine Operation Handbook, 2017
The occurrence of positive tests during product sterility testing should be investigated to determine if the growth resulted from microorganisms surviving the sterilization process. Any growth should be subcultured and identified, and the organism(s) should be compared to those derived from environmental monitoring or bioburden determinations. The laboratory performing the test should review all procedures and precautions taken as a part of their program to minimize false positives. Examine the lab’s false positive rate. The industry average is 0.1%. The lab should indicate if the test sample packaging is compromised in any way prior to testing. If so, the product should not be tested. If there is no indication that a deviation from procedure has occurred during the handling and testing of the device, then the growth must be considered a true positive. Precautions, such as the following, can be taken by the manufacturer to minimize false positives: Assure the product sample is small enough to fit within the lab’s largest container. The fewer the manipulations required during the test, the better.If the product is large and complex, subdivide it in such a manner that the subsequent pieces are easily transferred to the culture medium.Transfer test samples after exposure to the sterilization process in a plastic bag to reduce contamination that may accumulate on the outside of the package during shipping.
Surface modification of PHBV nanofiber mats for rapid cell cultivation and harvesting
Published in Journal of Biomaterials Science, Polymer Edition, 2018
Young-Gwang Ko, Young-Jin Kim, Won Ho Park, Donghwan Cho, Ho Yun Chung, Oh Hyeong Kwon
ADSCs were separated and subcultured. A piece of human adipose tissue was rinsed with phosphate buffered saline (PBS) containing an antibacterial agent, and then chopped. This tissue construct was shaken in 0.06% collagenase type I solution (Sigma, U.S.A.) in an incubator (37 °C, 5% CO2) for 12 h. The treated mixture was centrifuged, and the precipitated tissue portion was distributed in alpha minimum essential medium (α‐MEM, Gibco BRL, U.S.A.) containing 10% fetal bovine serum (FBS, Gibco BRL, U.S.A.) and 1% penicillin G-streptomycin (PGS, Gibco BRL, U.S.A.). Finally, the cell suspension was filtrated with a 70-μm pore filter (BD Bioscience, Bedford, U.S.A.) and cultured in an incubator. The cells were subcultured two times after reaching at least 90% confluence on the culture plate surface.