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Kinetic oxygen measurements by CVC96 in L-929 cell cultures
- Ulrich Plate†1Email author,
- Tobias Polifke†2,
- Dieter Sommer†1,
- Jörg Wünnenberg†1 and
- Hans-Peter Wiesmann†
© Plate et al; licensee BioMed Central Ltd. 2006
Received: 21 November 2005
Accepted: 01 March 2006
Published: 01 March 2006
Generally animal and human cells use oxygen during their whole life. Consequently the oxygen use is a simple indicator to test the vitality of cells. When the vitality decreases by the delivery of toxic substances the decrease can be observed directly by the oxygen-use of the cells. To get fast information of the vitality of cells we have measured the O2-tension by testing a new model of a bioreactor, the Cell Vitality Checker 96 (CVC96), in practical application. With this CVC96, soon a simple test will exist for the measurement of the oxygen use. In this respect the question had to be answered whether the use in the laboratory is easy and whether oxygen as a parameter in the vitality test can also be applied in future for problems in the field of material testing.
A recent challenge to bone and cartilage tissue engineering is to lift up research-scale products to a level of reproducible tissue substitute fabrication that is clinically effective, e.g. by the use of bioreactors [1, 2]. Bioreactors can be considered as devices in which biological and/or biochemical processes are performed under controlled conditions (e.g. pH, temperature, pressure, oxygen supply, nutrient supply and waste removal). Most of the bioreactors were initially developed to test biomaterials . The survival of cells in vivo as well as in bioreactors depends on the response of the distinct cells to the environment. A higher oxygen tension for example is needed for osteoblastic differentiation, whereas prolonged hypoxia favours formation of cartilage or fibrous tissue . The adjustment of oxygen tension in bioreactors is therefore a critical aspect in bioreactor design. The simplest and most widely used bioreactor for bone and cartilage tissue engineering today is indeed the culture dish . Animal and human cells use oxygen during their whole life. Consequently the oxygen use is a simple indicator to test the vitality of cells.
Vitality and cytotoxicity tests are established techniques in different fields such as cell culture control, search of active agents and also in other medical techniques. To the broad field of medical technology belongs also the testing of biocompatibility in the field of development and examination of tooth- and bone replacement tissues in cranio-maxilliofacial surgery. The research group for biomineralization and tissue engineering of this department carries out the testing of biocompatibility.
DSMZ Cell Culture Data for L-929 cells.
mouse connective tissue fibroblast
established from the normal subcutaneous areola and adipose tissue of a male C3H/An mouse; used as target in TNF detection assays
fibroblasts growing as monolayer
90% RPMI 1640 + 10% FBS
split confluent cultures 1:5 to 1:10 using trypsin (do not use trypsin/EDTA); after 2–3 days monolayer will be confluent; split 2–3 times a week; seed out initially at about 1.0–2.0 × 106 cells/25 cm2
at 37°C with 5% CO2
ca. 21–24 hours
ca. 4–8 × 106 cells/25 cm2
frozen with 70% medium, 20% FBS, 10% DMSO at about 1–2 × 106 cells/ampoule
negative in DAPI, microbiological culture, PCR assays
confirmed as mouse with IEF of AST, MDH, PEP B
murine hypertriploid karyotype – 61–67, 12–16 centric fusion markers present
ELISA: reverse transcriptase negative
The L-929 cell cultures were seeded in 96-wells. After becoming confluent, the cells were treated with three different types of media: a) media that had been incubated for 10 days with PMMA (poly-methyl-methacrylate), a preferable inert material for applications in medical technology, b) media that had been incubated for ten days with a collagen matrix, a material known to support the growth of bone cells. Besides the differently treated cells also untreated cells were measured as a reference. Further glutaraldehyde, a toxic chemical substance, was added to the L-929 cell cultures in different concentrations (0.1%, 1.0%, 10%). The cells were kept at 37°C in CO2-incubators during the whole experimental time and were removed from the incubator only for measurements. The measurements were carried out in a fluorescence-reader, FLX800T instrument from Bio-Tek Instruments GmbH, with which the data could be read in discontinuous kinetic files. The same modified media with PMMA and collagen were also tested for cell vitality in an identical attempt with following evaluation by a Resazurin-assay (CCS-PRINCESS® CELIA Instant Cytotoxicity-Assay 2004).
Results and discussion
The differences between the influence of collagen matrix, which showed as to expected a clear stimulating effect on the cells and that of PMMA, which showed no differences to the untreated medium, is clearly visible (Fig. 2, bottom). Also for this question clear results can be observed after 5 to 10 hours.
The usage of the new CVC96-assays is easier in relation to the standard assays, because the pipetting work steps are not necessary. The quality of the data and their reliability increase by kinetic evaluation. The experiments can be carried out in shorter time, because in many cases no standardized temporal end-point of the measurement has to be awaited.
Up to now only the results of first praxis tests could be shown. Surpassing this first practical test in future, the validity of the data in relation to standard tests must be intensively investigated so that afterwards fundamental questions of biocompatibility can be investigated for pure and mixed materials. We regard CVC96 as an interesting assay which allows new possibilities of investigations on the basis of kinetic observations and gives new insights and faster results. Thus it should be extensively validated after being brought onto the market by the producer. This will include statistical relevant testing with different kind of cells and a greater number n of tested material.
This project is supported by grant WI 1769/1-2 from the Deutsche Forschungsgemeinschaft.
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