the determination of cytotoxicity is part of the initial evaluation process stipulated by iso standards . however , it is not only the absence of a toxic effect that describes the term biocompatibility , but also the presence of a positive influence in terms of biofunctionality ( e.g. , promotion of wound healing ) . cell culture systems may be of value in testing the biocompatibility of drugs , biomaterials or treatment techniques , as they allow the direct measurement of cytotoxicity and effect on cellular growth or the determination of tissue / material interactions . at present , a wide variety of self - initiated and commercially available test methods are used . these include different types of cell lines , such as cultured fibroblasts from human skin , buccal mucosa , periodontal membrane , embryonic lung , epithelial and hela cells , cultures of human keratinocytes and hacat cells , different murine cell lines ( c3h - l , balb / c 3t3 , l929 , liver and spleen cell lines , others ) , t - lymphocytes from lymph nodes and macrophages obtained by lavage . all of these cells are suitable for biocompatibility tests . nevertheless , the general opinion is that toxicity tests in vitro are more convincing when performed with cells that are homologous with the human tissue concerned . in accordance , appropriate cell lines for use in cytotoxicity and tolerance tests concerning the skin would be human dermal fibroblasts and human epidermal keratinocytes . 1 ) ) . their main function is the maintenance of the structural integrity through continuous secretion of macromolecules of the extra - cellular matrix ( collagen , glycosamine glycanes and glycoproteins ) . , fibroblasts can be isolated from skin biopsies or phimoses through enzymatic digestion of the dermis or by explant method . they are available as the primary cell line nhdf ( normal human dermal fibroblasts ) from promocell ( heidelberg , germany ) . keratinocytes are the most common cell type of the epidermis ( figure 1 ( fig . they develop from epidermal stem cells located in the stratum basale ( lowest layer of the epidermis ) and differentiate into corneocytes during their movement to the stratum corneum ( upper layer of the epidermis ) . there , they form a barrier which protects the body from foreign bodies and germs as well as dehydration . keratinocytes take an active part in the immune response , inflammatory processes , and wound healing as they are able to synthesize a broad range of cytokines , growth factors , and complement factors . like fibroblasts , they can be isolated from skin biopsies and phimoses through enzymatic treatment of the epidermis . promocell ( heidelberg , germany ) also offers a primary keratinocyte cell line ( nhek , normal human epidermal keratinocytes ) . after a few passages , the cells lose their proliferation ability , probably due to chromosomal alterations . those developing changes can influence experiment outcomes , which means there is a relatively small time frame for repeating tests . hacat cells are spontaneously immortalized keratinocytes . they originate from the isolate of the distal periphery of a melanoma located on the upper half of the back of a 62-year - old male patient and have been described in detail by boukamp et al . . while they demonstrate a transformed phenotype in vitro , they are not tumorogenic , exhibit a steady expression of specific keratins and other identification markers , and show normal differentiation . fusenig , dkfz , heidelberg , germany ) are cultured in dulbecco s modified eagle s medium ( dmem , promocell , heidelberg , germany ) , supplemented with 10% fetal calf serum ( fcs , promocell , heidelberg , germany ) and 1% antibiotic - antimycotic mix ( psf , promocell , heidelberg , germany ) at 37c in a humidified atmosphere containing 5% co2 . primary fibroblasts ( nhdf , promocell , heidelberg , germany ) and keratinocytes ( nhek , promocell , heidelberg , germany ) can be maintained in appropriate basal media supplemented with growth factors but without antibiotics under the same conditions . for experiments , the cells are seeded at the required density into 96-well plates , 12-well plates , object slides etc . after allowing the cells to attach to their new surface therefore , the cell culture medium is replaced by either fresh culture medium ( negative control / medium control ) or the test substance dissolved in culture medium or the material extract prepared according to the din standard en iso 10993 - 12 . the subsequent assessment of the in vitro cytotoxicity of the substance is often a qualitative analysis based on the morphological examination of cell damage and growth after direct or indirect contact with the material . in general , methods involving the measurement of cellular growth ( cell counting or confluency evaluation ) are more sensitive and discriminating than those in which the test materials are placed directly in the cell cultures to determine the zone of growth inhibition . however , dead cells lose their membrane integrity and are stained blue . this method is inexpensive and easy to perform , but unsuitable for high - throughput screening ; it is only recommendable for suspension cells , as adherent cells need to be trypsinized prior to counting . determination of cell proliferation and cell viability has become the key technology to assess the growth behavior in a cell culture . they are based on the determination of nucleic acids , metabolic activity , and protein content , or membrane integrity . a small selection ( table 1 ( tab . 1 ) ) , such as pico green dna cell proliferation assay , atplite luminescence atp detection assay , bc assay : protein quantitation kit , alamarblue proliferation assay and live / dead staining with syto-13 and ethd-2 , is discussed here . picogreen ( molecular probes inc , or , usa ) is a reagent for quantifying dsdna , which provides a stable measurement parameter . the relative fluorescence units measured correlate with the number of cells present ( figure 2 ) . cell number and picogreen fluorescence exhibit a linear relationship for low and medium cell densities , but which is lost for high cell densities . on the other hand , the assay shows a high sensitivity for low cell numbers , as it is able to detect dsdna concentrations as low as 25 pg / ml . equally sensitive is atplite ( perkin elmer life sciences , ma , usa ) , an atp monitoring system based on the detection of light generation by firefly luciferase . in an atp - dependent reaction , the amount of emitted light is directly proportional to the atp concentration ( figure 2 ( fig . atp is present in all metabolically active cells and declines rapidly when cells undergo necrosis and apoptosis . however , it allows only the measurement of viable cells and not the quantification of the total cell number . another possibility is the determination of the protein content , which naturally corresponds directly to the cell number ( figure 2 ( fig . the bc assay ( uptima interchim , france ) is derived from the biuret reaction ( cu + e  cu ) using bicinchoninic acid . it is based on the measurement of the formation of a water soluble , purple - colored complex of bicinchoninic acid and cu . the test is sensitive , but not as sensitive as the picogreen or atp assay , and low cell numbers do not provide the necessary protein concentration . in contrast to the first three methods , the alamarblue proliferation assay ( biosource , ca , usa ) is a non - destructive method . the test uses the indicator dye resazurin , which is dark blue in color and possesses little intrinsic fluorescence until it is reduced by the metabolic activity of viable cells to resorufin , which is pink and highly fluorescent . non - viable cells rapidly lose their metabolic capacity , do not reduce the dye , and thus do not generate a fluorescent signal . the assay allows simultaneous incubation and measurement but requires precisely defined time and temperature conditions , as the resorufin will continually develop . 3 ) shows a cell standard curve incubated with alamarblue measured after 3 h ( figure 3 a ( fig . all of the cell quantification methods discussed have advantages and disadvantages ( summarized in table 1 ( tab . 1 ) ) , and the most suitable test method for a given study must be chosen . besides , it is often necessary and always recommendable to verify the test results with at least two different assays . in that way , it is possible to exclude outside influences or false interactions of the components . the assays can be used to determine the cell proliferation over a particular time period ( figure 4 ( fig . so far , we have been able to demonstrate proliferative as well as cytotoxic effects of various test substances on keratinocytes and fibroblasts . cell proliferation tests are able to express toxic effects via loss of proliferation ability or reduction of living cells . these negative effects can be quantified by methods that determine cytotoxicity through the measurement of necrosis or apoptosis ( table 1 ( tab . 1 ) ) . staining with syto-13 and ethidiumhomodimer-2 enables the direct count of viable and dead cells . syto-13 and ethidiumhomodimer-2 ( molecular probes inc , or , usa ) are green and red fluorescent dyes that bind to nucleic acids . while syto-13 can permeate membranes and is therefore able to stain living cells , ethidiumhomodimer-2 can not , and can only enter dead cells , but possesses a higher binding affinity to dna than syto-13 . treatment of the cells with a toxic substance leads to a reduction in the ratio of green to red cells ( figure 7 ( fig . lactate dehydrogenase ( ldh ) in the supernatant is a sign for necrotic degradation of the cells depending on the release of ldh from the cytosol after cell membrane damage . the cytotoxicity detection kit ( roche , in , usa ) is a colorimetric assay which measures the activity of released ldh . cytotoxic substances which affect membrane integrity cause a rapid release of ldh compared to the medium control ( figure 8 ( fig . this process is accompanied by a rapid decline of cell viability ( figure 8 ( fig . this method is quick and easily adaptable for different types of cells and test conditions , but the assay is sensitive to interference from compounds with inherent ldh activity , such as serum - supplemented culture media . frequently , the treatment with a substance does not kill the cells directly , but induces cellular damage that subsequently leads to pathological events . the members of the caspase family play a key effector role in mammalian cell apoptosis . the caspase - glo 3/7 , 8 and 9 assays ( promega gmbh , mannheim , germany ) provide specific luminogenic substrates that form a luminescence signal proportional to the amount of caspase activity present ( figure 9 ( fig . the release of inflammatory cytokines accompanies the reaction of the cells to the test substance ( figure 10 ( fig . 10 ) ) . cells do not function independently from each other , they communicate through the secretion of interleukins such as il-6 and il-8 . interleukin 6 is a cytokine that provokes a broad range of cellular and physiological responses , e.g. inflammation , haematopoiesis and neuronal differentiation . interleukin 8 is a member of the superfamily of chemokines and acts as a chemo - attractant for neutrophils ; it plays a crucial role in many inflammatory events . the cytokine concentration in the supernatant can be easily measured by elisas specific for il-6 and il-8 ( milenia biotec , bad nauheim , germany ) . materials and substances used , for example , in the treatment of wounds do not only influence cell proliferation and viability , but also affect their migration capacity . fibroblasts and keratinocytes enable the maintenance of the barrier function of normal skin . upon wounding , this shield is disrupted and fibroblasts and keratinocytes migrate into the wound area to regenerate the skin . therefore , the cells must dissociate from one another and break the cell - matrix contacts to detach from the basal lamina . migration into the wounded area entails remodelling of the extra - cellular matrix by its proteolytic degradation as well as by de novo synthesis and deposition of newly formed matrix components . both degradation and reformation of the matrix are functions performed by dermal fibroblasts and epidermal keratinocytes . the mechanical scratch wounding of confluent monolayers ( figure 11 ( fig . 11 ) ) serves as a model to study cell migration at the wound margins . the method allows the direct measurement of cell migration and layer regeneration . using the scratch apparatus described by bth et al . so far , all featured test methods utilize cell monolayers which are an exclusively in vitro condition . evidence suggests that cells behave differently within these two dimensions than in their familiar 3-dimensional in vivo environment . for instance , fibroblasts reach confluency in monolayer cultures with a high proliferation rate and display protein biosynthesis levels that do not resemble the in vivo situation . on the other hand , fibroblasts embedded in 3-d collagen gels exhibit lower biosynthetic activity , regulated growth , and stellate shape ; they also have protruding dendritic extensions and a state of differentiation close to the in vivo situation . to mimic the conditions which cells experience in vivo , the use of 3-d cell cultures is becoming increasingly popular . ordered collagen matrices  ( orc ) use the long range assembly properties of type i collagen monomers , and the cells are seeded afterwards into the collagen gel formed . ( figure 12 ( fig . 12 ) ) is formed as a tissue - like structure from a collagen gel contracted by fibroblasts . with 3-d cultures it is possible to study collagen degradation as well as polymerization and deposition of newly synthesized collagen . they allow the study of tissue remodelling in vitro and provide an assay system for wound contraction . it is even possible to prepare skin - equivalent tissue by co - culturing with keratinocytes . of course , these are time - consuming and elaborate preparations that require operator experience and the appropriate evaluation methods . it has been reported that the use of physical wound treatment techniques , such as wira ( water filtered infrared a ) and hvpc ( high - voltage pulsed current ) , have a positive effect in terms of accelerated wound healing   or additional antibacterial activity . for the measurement of the effect of these physical methods in vitro or for the application of a new plasma device on cells , several considerations must be taken into account to design an appropriate study . for one , it is necessary to scale the in vivo application parameters to the in vitro use on cell monolayers ( figure 13 ( fig . 13 ) ) . for assessment of the bactericidal activity of hvpc , cotton pieces were soaked with bacterial suspension , placed on a steel plate , and covered with the electrode ( 42 ma , 128 hz , 30 min ) . cells need stable culture conditions that guarantee the establishment of an appropriate cell environment during the experiment . therefore , in vitro studies that have been performed to assess the bactericidal activity of hvpc can not be applied one - to - one on human cell cultures . in conclusion , for testing physical treatment methods , the choice will probably tend to the use of a 3-d culture system which more closely resembles the in vivo situation , although cellular effects are far easier to detect and quantify in cell monolayers than in 3-d cultures . hopefully , this summary of methods for measuring cell and tissue compatibility will provide ideas for the design of conclusive in vitro studies .
biocompatibility is one of the main requirements for the safe use of medical devices . 
 determination of cytotoxicity is part of the initial evaluation stipulated by iso standards for the biological evaluation of medical devices . 
 the use of cell cultures to test the biocompatibility of drugs , biomaterials or treatment techniques used in various disciplines is gaining in importance . 
 a wide variety of self - initiated and commercially available cell lines has been evaluated and used : cultured fibroblasts from human skin , buccal mucosa , periodontal membrane , embryonic lung , epithelial and hela cells ; cultures of human keratinocytes and hacat cells ; different murine cell lines ( c3h - l , balb / c 3t3 , l929 and others ) as well as murine cells cultured from liver and spleen ; t - lymphocytes from lymph nodes and macrophages obtained by lavage.all of the above cells are suitable for use in biocompatibility tests . nevertheless , the general opinion is that toxicity tests in vitro will be more convincing when performed with cells that are homologous with the human tissue concerned . in accordance , 
 appropriate cell lines for use in cytotoxicity and tolerance tests concerning the skin would be human dermal fibroblasts and human epidermal keratinocytes , as they take an active part in the immune response , inflammatory processes , and wound healing.the evaluation of the in vitro cytotoxicity of a biomaterial is often a qualitative analysis based on the morphological examination of cell damage and growth after direct or indirect contact with the material . 
 different commercial assays based on the determination of nucleic acids , metabolic activity , protein content or membrane integrity are available to measure cell proliferation and cell viability . a small selection 
 pico green dna cell proliferation assay , atplite luminescence atp detection assay , bc assay : protein quantitation kit , alamarblue proliferation assay and live / dead staining with syto-13 and ethd-2  are discussed concerning sensitivity , reliability and applicability .