In vitro activation of murine bone marrow-derived macrophages with cisplatin and mitomycin-C

Peritoneal macrophages from BALB/c mice after treatment for 24 h in vitro with cisplatin, lipopolysaccharide (LPS) or mitomycin-C were rendered significantly cytotoxic against L-929 tumor target cells. In a similar experiment none of these agents could induce tumoricidal activity of fresh non-adherent bone marrow cells (NABMC). NABMC when incubated in medium alone or in medium containing L-929 culture medium (L-929 CM), a form of macrophage colony stimulating factor (M-CSF), for three days matured to macrophages which were positive for non-specific esterase staining. These bone marrow-derived macrophages cultured with medium alone did not respond to cisplatin. LPS or mitomycin-C for induction of tumoricidal activity whereas bone marrow derived macrophages with that were incubated with L-929 CM showed also significantly enhanced cytotoxicity after treatment with cisplatin, LPS and mitomycin-C. Culturing of NABMC with L-929 CM significantly enhanced cell survival as compared to the cells incubated in medium alone. These results suggest that bone marrow cells not only mature in L-929 CM but also are primed by L-929 CM for induction of tumoricidal activity.     

A specific and reliable bioassay for the detection of femtomolar levels of human and murine tumor necrosis factors

A reliable, highly sensitive, cytolytic bioassay for the quantitation of both human and murine tumor necrosis factor (TNF) is described. The assay is 2-180-fold more sensitive than other currently described bio- or immunoassays (limits of detection: 500 fg/ml (29 fmol/l) human TNF-alpha, 200 fg/ml (12 fmol/l) murine TNF-alpha and 130 fg/ml (7 fmol/l) human TNF-beta). The assay, which uses L929-8, a newly isolated subclone of the murine fibroblastoid cell line L929, detects human TNF-alpha approximately 180-fold more sensitively than previously described L929 subclone assays. Maximum sensitivity is obtained by preincubating L929-8 cells at 37 degrees C with 2 micrograms/ml actinomycin D (1-2 h), then culturing with TNF at 40 degrees C for 20 h in medium containing high serum (15% FBS). Relative viable cell content in 96-well microtiter plates is determined colorimetrically by uptake of the non-carcinogenic dye neutral red. Other cytokines have no effect, either alone or in combination with TNF. Cytokines tested were IL-1 through IL-6, GM-CSF, G-CSF, CSF-1, LIF, TGF-beta, NGF, Epo or IFN-gamma, LPS, PGE2, dexamethasone and cyclosporin A, also have no effect, either alone or in combination with TNF. L929-8 cells maintain the above sensitivity to TNF for at least 4 months in continuous culture. Thus, the assay allows rapid, inexpensive, reliable and specific quantitation of rodent and human TNFs. Its very high sensitivity should allow accurate detection of biologically active TNF in biological fluids such as human serum.     

Interferon induces an antiviral state in ganglioside-deficient transformed mouse fibroblasts

The antiviral activity of mouse fibroblast interferon against vesicular stomatitis virus was investigated in L-929 mouse fibroblasts and the ganglioside-deficient L-929 mutant cells (ATCC clone NCTC 2071). Although it has been widely reported that gangliosides serve as primary receptors for interferon at the cellular membrane, only a small difference in interferon sensitivity was observed between the wild-type L-929 and the ganglioside-deficient NCTC 2071 cells. It was not possible, however, to overcome this difference by administration of exogenous gangliosides.     

Replication of sialodacryoadenitis virus in mouse L-2 cells

Summary Sialodacryoadenitis (SDA) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (SDAV). The study of SDAV has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. The purpose of this study, therefore, was to compare the ability of SDAV to replicate in the permanent cell lines, LBC, of rat origin, and the mouse cell lines. L-929 and L-2. Following 2 to 6 repeated passages of SDAV in LBC cells, the virus could be readily propagated in LBC and L-2 cells, but not in L-929 cells. Similarly, SDAV adapted to replicate directly in L-2 cells could be readily propagated in LBC, but not L-929 cells. In LBC and L-2 cells, cytopathic effect (CPE), viral antigen, viral particles, and virus infectivity could be demonstrated. Titers of up to 10^8.0 infectious viral particles/0.25 ml of culture fluid were obtained at 48 hours in L-2 cells. Titers in LBC cells were one to two logs lower. When susceptible rats were inoculated with eighth passage L-2 cell-adapted virus, they developed typical lesions of SDA. Virus could be recovered from infected tissues and propagated in L-2 cells on first passage. The ability to propagate SDAV to high titers in the widely available L-2 cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses.     

Mechanisms of cytotoxicity of nickel ions based on gene expression profiles

This study investigated cytotoxic effects of Ni(II) to mouse fibroblast cells (L-929) on the level of gene expression profiles with cDNA microarray. The gene expression profiles of L-929 were detected after the cells were cultured in the medium with 200 microm Ni(II) for 24, 48 and 72 h, respectively, and the cytotoxicity of Ni(II) was evaluated with methylthiazoltetrazolium (MTT) assay. 20 up-regulated genes and 19 down-regulated genes were differentially expressed in all three-culture periods. Gene ontology analysis showed that the L-929 cells which responded to Ni(II) covered a broad range of functional gene groups including cellular biological process, molecular function, and cellular component. Ni(II) has extensive effects on cells by inhibiting cell proliferation and differentiation through inducing cell apoptosis, affecting cell development and influencing cholesterol metabolism.     

Immunological and biochemical characterization of biglycan-like haemopoietic factor.

Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L-929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N-glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells.     

A comparison between two methods for measuring tumor necrosis factor in biological fluids

The current study was undertaken to compare two methods for the efficiency of measuring tumor necrosis factor (TNF-α) in biological fluids, which is species undependent, reliable, sensitive, simple and not expensive. We have compared the MTT tetrazolium cytotoxic assay [1,2] and the³H-thymidine (³H-TdR) incorporation cytostatic assay for measuring the anti-tumor activity of human recombinant TNF-α, of human colonic tissue and of supernatants ofin vitro stimulated human and rat peritoneal macrophages. Two target cell-lines, namely murine myelomonocytic leukaemia WEHI-164- and L-929-transformed murine fibroblast cell-lines, were used in the MTT assay. The L-929 line was also used in the³H-TdR assay. WEHI-164 was more sensitive than the L-929 cell-line in the MTT cytotoxic assay. Furthermore, the MTT assay was more sensitive to TNF-α than the³H-TdR assay. Both methods can be used for the detection of anti-tumor activity in biological fluids but the MTT cytotoxic method has the advantage of being more sensitive and more simple.

     

The Cytotoxic Effect of Mouse Macrophages Stimulated in Vitro by a β-1,3-d-Glucan from Yeast Cell Walls

Macrophages stimulated by an insoluble β-1,3- d -glucan from yeast cell walls were able to destroy tumour cells as measured hy the release of radioactive label from prelabelled ¹⁴C-thymidine cells. Target cells were B-16 melanoma. P-815 rrmstocytoma, and the L-929 cell line, A significant target cell killing by macrophages stimulated by glucan was observed after 72–96 h. The cytolysis of L-929 cells was investigated in some detail. No stable soluble cytolytic factor appeared to be released into the medium during the stimulation of macrophages by glucan. since cell-free spent medium had no cytotoxic effect on L-929 cells. The densities of the macrophage monolayers were critical for an effective target cell killing; dense cultures showed more cyioioxicity than less dense cultures. The kinetics of the development of macrophagc-mediated cytotoicit suggests a minimum stimulation period of 4 days for maximal cylolysis.     

三种比色法评价4种牙科金属材料对L-929细胞的毒性

背景：采用不同方法评价材料的细胞毒性可能会得出不同的实验结果。 目的：采用3种比色法评价镍铬合金、钴铬合金、3铬13及纯钛等牙科金属材料对小鼠成纤维细胞(L-929细胞)的细胞毒性。 方法：以镍铬合金、钴铬合金、3铬13及纯钛4种牙科金属材料的浸提液分别作用于体外培养的L-929细胞24,72 h。以体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阴性对照组,以0.7%丙烯酰胺+体积分数10%小牛血清+高糖DMEM培养液培养的L-929细胞为阳性对照组,分别采用MTT、CCK-8及结晶紫3种比色法检测上述材料的细胞毒性。 结果与结论：①4种材料浸提液中培养的细胞形态正常,胞内结构清晰,随着培养时间延长细胞大量增殖,与阴性对照组细胞形态无明显差异。阳性对照组细胞数量明显减少,形态完整性受破坏,形成大量细胞碎片。②培养24 h时,CCK-8比色法检测中钴铬合金组的细胞相对增殖率低于阴性对照组(P < 0.05),MTT及结晶紫比色法检测中钴铬合金组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05);培养72 h时,MTT比色法检测中4种牙科金属材料组细胞相对增殖率低于阴性对照组(P < 0.01),CCK-8及结晶紫比色法检测中4组的细胞相对增殖率与阴性对照组比较差异无显著性意义(P > 0.05),但材料细胞毒性均为0-1级。表明上述4种牙科金属材料细胞毒性均在临床应用的允许范围内,具有良好的生物安全性。     

Serum-free in vitro bioassay for the detection of tumor necrosis factor

A sensitive, reproducible in vitro bioassay is described for quantitating the cytolytic activity of tumor necrosis factor (TNF). The assay target cells, murine connective tissue L-M, are propagated and the assay performed under serum-free conditions. The quantitation of cytolytic activity is based on the ability of TNF to lyse L-M cells in the presence of actinomycin D, as measured by crystal violet dye uptake of residual viable cells. The assay is sensitive to 88 pg/ml TNF-alpha. The simplicity of the culture medium combined with high sensitivity and low variability make this a particularly well-suited bioassay for routine detection of TNF cytolytic activity.     

Protection from tumor necrosis factor-mediated cytolysis by platelets.

Infiltrating macrophages elicit tumor-destructive reactions by releasing cytolytic factors including tumor necrosis factor alpha (TNF-alpha). Because platelets represent another major component of the cell infiltrate in tumors, we examined whether they could affect TNF alpha-induced cell death. Exposure of L-929 fibrosarcoma cells to human platelets reduced TNF alpha-induced cytotoxicity and cytolysis, as determined by 51Cr release assay and DNA fragmentation assay. This inhibitory effect, which depended on the concentration of platelets (0.1 to 10 x 10(6)/0.1 ml), was as high as 50%. The decrease in responsiveness to TNF-alpha reflected neither a degradation of TNF-alpha nor an inability of L-929 cells to bind TNF-alpha. Indeed, even though Scatchard analysis indicated the presence of 100 to 150 125I-TNF-alpha binding sites/platelet with a kd of 3.8 to 6.4 nM, addition of platelets up to 5 x 10(6)/0.1 ml did not compete with 125I-TNF-alpha binding to L-929 cells. Furthermore, addition of platelets 1 or 2 hours after that of TNF-alpha was still protective suggesting that platelets rather promoted hyporesponsiveness of L-929 cells to a postbinding effect of TNF-alpha Platelet-induced reduction of TNF-alpha response could be reproduced with supernatant fluids from platelets incubated at 37 degrees C for 24 hours. The platelet-derived factor responsible for this effect was found to be a lipid of low molecular weight with high affinity for albumin and charcoal. A role for 12(S) hydroxyeicosatetraenoic acid is proposed because this metabolite reduced TNF-alpha-induced cytolysis in a dose-dependent manner, whereas other platelet-derived lipids including thromboxane A2 and platelet activating factor were inactive. These observations indicate that the role of associated platelets has to be considered when analyzing the cytotoxic and cytolytic activity of macrophage-derived TNF-alpha on tumor cells.     

The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.     

Rabbit tumor necrosis factor: mechanism of action.

Rabbit tumor necrosis factor (TNF) was examined for effects on normal and transformed cells in culture. Several assays for killing of L-929 cell targets were developed, and their sensitivities were compared. Normal cells were not killed by TNF, and the discrimination between normal and transformed cells was shown not to be due to a cell cycle-dependent mechanism. TNF killing of L-929 cells was delayed for 10 to 12 h and thereafter showed concentration and time-dependent increases in cytolysis. Actinomycin D or cycloheximide treatment of L-929 cells resulted in an enhancement of the rate of cell killing as well as a shortening of the preceding lag period. TNF killing of L-929 cells was temperature dependent; cells were considerably more resistant to lysis at 25 degrees C and showed enhanced killing at 39 degrees C as compared to 37 degrees C controls. The slope of the dose curve showed less than single-hit kinetics. A model for cell killing whose general features incorporate both the specificity and catalytic properties of an enzymatic reaction is proposed for TNF action.     

时间分辨荧光分析测定细胞活性和破膜法实验研究

采用Eu3+-DTPA标记L-929株靶细胞，经时间分辨荧光仪测定，建立一种崭新的时间分辨荧光分析法来测定细胞活性，此法具有灵敏度高，有效期长，不污染环境等优点。     

Bluetongue virus propagation and plaque assay: variation due to medium and serum supplement

Two base media, minimal essential medium (MEM) and RPMI 1640, were supplemented with a variety of serum extenders and/or substitutes for the purpose of defining a medium formulation capable of supporting good cell (VERO) growth and virologic assays. Bluetongue virus (BTV), the prototype Orbivirus in the Reoviridae, was used in all studies. In general, VERO cells grown in RPMI performed better than those grown in MEM relative to cell growth, virus production and plaque assay. RPMI was better for supporting cell growth when serum extenders (NuSerum or SerXtend) were employed as supplements. Relative to virologic techniques, cells grown in RPMI produced higher virus titers in both propagation studies and plaque assays. The interval from infection to greater than 90% cytopathic effect (CPE) was consistently shorter with RPMI as the base medium. Cell cultures supported with RPMI base medium, supplemented with 3.5% FBS with SerXtend, provided the best overall performance relative to: (a) amount of virus produced by infected cell monolayers, (b) sensitivity to productive infection under overlay conditions (revealed the highest titer of a standard virus stock) and (c) plaque assay quality including cell quality, plaque size and plaque clarity.     

In vitro proliferation and differentiation of human mesenchymal stem cells cultured in autologous plasma derived from bone marrow

Expansion of human mesenchymal stem cells (hMSCs) in medium supplemented with fetal bovine serum (FBS) has a potential risk of transmitting viral and prion diseases and causing immunological rejection. The aim of our present study was to find a substitute for the traditional FBS in culture of hMSCs to facilitate the clinical application of hMSCs. We used autologous plasma derived from bone marrow (APM) as a substitute for FBS and found that, when cultured with APM, the cell surface markers and the proportion of hMSCs in the G(0)/G(1) phase and the S+G(2)/M phase resembled those cultured with FBS. However, there were fewer early apoptotic cells in APM-supplemented medium than in FBS (p < 0.01). APM resulted in much greater thymidine incorporation than FBS (p < 0.001). There were significantly more alkaline phosphatase (ALP)-positive fibroblast colony-forming units (CFU-Fs) covering larger areas in APM than in FBS (p < 0.01). Also, APM induced greater ALP activity, more mineralized nodules, and greater expression of osteogenic genes than did FBS. In addition, when cultured in adipogenic medium, there were fewer oil-red O-positive cells and lower expression of adipogenic gene with APM than with FBS. In conclusion, expansion of hMSCs in APM-supplemented medium instead of traditional FBS is more advantageous. It could promote cell proliferation, enhance osteogenic differentiation, and suppress adipogenic differentiation of hMSCs and is therefore a safer and more effective substitute for FBS in clinical cytotherapy processes.     

Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage

Analysis of cryopreserved peripheral mononuclear cells (PBMC) is important for evaluating new vaccines in immune based therapies and in pathogenesis studies. To ensure comparable assay results from different laboratories and points of time, collaborative research in multicenter trials needs reliable and reproducible cryopreservation protocols that maintain cell viability and functionality. Current cryomedia consist largely of fetal bovine serum (FBS), a natural mix of growth factors, cytokines, and undefined compounds. Standardized procedures are not possible, as FBS can affect the antigen-specific T-cell response, the most important parameter in functionality assays. Also, worldwide sample exchange is complicated by the strict import restrictions on FBS, because of transfection risk. After establishing a serum-free cryopreservation protocol that maintains cell viability, recovery and antigen-specific T-cell response of PBMC comparably to FBS-based cryomedia (Germann et al., 2011), the aim of this study was the complete avoidance of animal proteins and products in combination with efficient cryopreservation. As long-term stability of the cryopreservation process is crucial for retrospective evaluation of samples at different points of time, PBMC were analyzed after storage for maximal four weeks and again after approximately six months. The cryopreservation efficiency of the protein-free and fully chemically defined cryomedium was comparable to FBS-medium after storage for few weeks and several months. Directly after thawing, this medium yielded viabilities over 97% and recovery values over 84%. Also, the specific T-cell functionality was preserved. Additionally, short-term and six month cryopreservation gave comparable results. The fully chemically defined medium presented here will increase standardization and reproducibility of analysis in multicenter-studies or in retrospective evaluation.     

Optimization of artificial urine formula for in vitro cellular study compared with native urine

Several artificial urine (AU) formulas have been developed to mimic the normal urine. Most of them are protein-free, particularly when secreted proteins (secretome) is to be analyzed. However, the normal urine actually contains a tiny amount of proteins. We hypothesized that urinary proteins at physiologic level play a role in preservation of renal cell biology and function. This study evaluated the effects from supplementation of 0-10% fetal bovine serum (FBS) into the well-established AU-Siriraj protocol on MDCK renal tubular cells. Time to deformation (T_D) was reduced by both native urine and AU-Siriraj without/with FBS compared with complete culture medium (control). Among the native urine and AU-Siriraj without/with FBS, the cells in AU-Siriraj+2.5% FBS had the longest T_D. Supplementation of FBS increased cell death in a dose-dependent manner (but still <10%). Transepithelial electrical resistance (TER) of the polarized cells in the native urine was comparable to the control, whereas that of the cells in AU-Siriraj+2.5% FBS had the highest TER. These data indicate that supplementation of 2.5% FBS into AU-Siriraj can prolong time to deformation and enhance polarization of renal tubular cells. Therefore, AU-Siriraj+2.5% FBS is highly recommended for in vitro study of cell biology and function (when secretome is not subjected to analysis).     

骨髓间充质干细胞-海螵蛸复合支架的细胞毒性评估

文题释义：骨髓间充质干细胞复合海螵蛸支架：以中药海螵蛸为载体支架,骨髓间充质干细胞为种子细胞,二者共同培养构建复合生物支架,经过各项生物安全性测评后应用于骨缺损的治疗。制作过程为：选用第3代骨髓间充质干细胞,经0.25%胰酶消化制成细胞悬液,将细胞浓度调整为5×10⁸ L^-1,用移液枪将骨髓间充质干细胞悬液缓慢逐滴滴加于24孔板中的海螵蛸上,200 μL/孔,分2次缓慢接种,尽量不使细胞从材料上溢出,置于培养箱中培养4 h,待细胞充分黏附于海螵蛸支架材料后,缓慢逐滴加入培养液700 μL,将支架与细胞复合培养8 d,两三天换液1次。细胞毒性评估：是采用受试物与细胞共同培养的方法,对样品进行毒理学风险评估。生物材料应用于体内后,可能与体内细胞的细胞膜、细胞器、蛋白质合成、DNA合成等相互作用,产生细胞毒性。根据ISO10993-5标准,在复合支架应用于临床治疗前,需通过复合材料浸提液试验以测定其对细胞活性、功能、遗传的影响。  摘要背景：作为骨组织工程的修复材料,应具有良好的生物相容性及降解吸收性,许多学者在此方面也做了较深入的研究,但中药复合细胞生物支架的研究较少。目的：参照《医疗器械生物学评价标准》,对兔骨髓间充质干细胞-海螵蛸生物复合支架进行体外细胞毒性检测,评估复合支架的毒性等级,为临床应用生物支架提供实验依据。方法：依照ISO标准按“材料面积∶浸提介质体积=3-6 cm²∶1 mL”制作骨髓间充质干细胞-海螵蛸生物复合支架材料浸提液。制备L-929细胞悬液,将细胞浓度调整至1×10⁷ L^-1进行接种培养,设置阳性对照组(含苯酚的DMEM培养液)、实验组(材料浸提液)、阴性对照组(新鲜DMEM培养液)。培养24,48,72 h采用MTT法检测L-929细胞吸光度值,计算各组细胞的相对增殖率,评估复合支架毒性等级。结果与结论：实验组、阴性对照组、阳性对照组吸光度值在不同时间点不完全相同(P=0.000 < 0.01),各时间点内比较,实验组与阴性对照组吸光度值均明显高于阳性对照组(P < 0.01);兔骨髓间充质干细胞-海螵蛸生物支架细胞毒性为1级。结果说明兔骨髓间充质干细胞-海螵蛸生物支架无明显毒性作用,符合生物材料应用要求。  ORCID: 0000-0002-0003-6228(彭雅) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

In vitro cytocompatibility studies of Diamond Like Carbon coatings on titanium

Diamond like carbon (DLC) films were deposited on to titanium (Ti) substrates by Plasma Enhanced Chemical Vapour Deposition (PECVD) process. The quality of the films were checked by Raman spectra and nano-hardness tests. The cytocompatibility of titanium and DLC coated titanium were studied using continuous cell lines of mouse fibroblast cells ( L-929), Human Osteoblast cells (HOS) and primary human umbilical cord vein endothelial cells (HUVEC). The cellular responses to the materials were assessed both quantitatively and qualitatively. The adhesion and spreading of cells on materials were compared using Ti as a control. Present study indicates an improved cytocompatibility of DLC coated Ti in comparison to bare Ti.