2 CHO ko cell line
CHO细胞无血清培养基技术手册
(1)无血清培养基，为一般意义上无血清培养基，用各类可替代血清功能的生物材料配制细胞培养基，如牛血清白蛋白(BSA)、转铁蛋白、胰岛素等生物大分子物质，以及从血清中提取的去除蛋白质的混合脂类以及水解蛋白等。其特点是培养基中的蛋白含量较高,添加物质的化学成分不明确，其中含有大量的动物来源蛋白。
5 CHO细胞无血清无动物组分培养基（SAF-CHO-G-004）
其实个性化培养基并不新鲜，在国外生物制药企业普遍采用个性化培养基，世界著名的生物制药公司（如Amgen、Genetech）往往和培养基企业合作，通过细胞培养基的客户定制方式，研制最适合自身细胞特点的个性化细胞培养基应用于生产实践，用于提高细胞产率和生产效率。另有数据显示，国际上的细胞培养基生产企业，个性化、定制培养基占其培养基业务的90%以上，目录培养基不足10%。
ATCC提供的CHO/dhfr-细胞照片
2 CHO细胞表达系统的优势与特点
由于哺乳动物细胞结构、功能和基因表达调控的复杂性，外源基因在哺乳动物细胞中的表达与在原核生物中的表达存在较大差异，因而外源基因在哺乳动物细胞中高效表达所需的元件也不同于在原核生物细胞中的表达所需的元件。外源基因在哺乳动物细胞中的表达包括基因的转录、mRNA翻译及翻译后蛋白质的加工等过程，如蛋白质的糖基化、磷酸化、寡聚体的形成以及蛋白质分子内或分子间二硫键的形成等比较复杂，要表达具有生物学功能的蛋白质如膜蛋白、抗体和具有特异性催化功能的酶需要在哺乳动物细胞中进行。
其它
无水葡萄糖、HEPES、丙酮酸钠、亚油酸、硫辛酸、胰岛素、亚硒酸钠、植物蛋白等
2008-Cytoprotective effect of fucoxanthin isolated from brown=FX=细胞保护
ORIGINAL PAPERCytoprotective effect of fucoxanthin isolated from brown algae Sargassum siliquastrum against H 2O 2-induced cell damageSoo-Jin Heo ÆSeok-Chun Ko ÆSung-Myung Kang ÆHahk-Soo Kang ÆJong-Pyung Kim ÆSoo-Hyun Kim ÆKi-Wan Lee ÆMan-Gi Cho ÆYou-Jin JeonReceived:26March 2008/Revised:9June 2008/Accepted:23June 2008/Published online:22July 2008ÓSpringer-Verlag 2008Abstract In this study,the cytoprotective effect of fuco-xanthin,which was isolated from Sargassum siliquastrum ,against oxidative stress induced DNA damage was investi-gated.Fucoxanthin,a kind of carotenoid,was pretreated to the medium and the protective effect was evaluated via 20,70-dichlorodihydrofluorescein diacetate,3-(4,5-dimeth-ylthiazol-2-yl)2,5-diphenyltetrazolium bromide,and comet assays.Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H 2O 2),but the production was significantly reduced by the treatment with fucoxanthin.The fucoxanthin strongly enhanced cell via-bility against H 2O 2induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner.Furthermore,a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye.These results clearlyindicate that fucoxanthin isolated from S.siliquastrum possesses prominent antioxidant activity against H 2O 2-mediated cell damage and which might be a potential ther-apeutic agent for treating or preventing several diseases implicated with oxidative stress.Keywords Fucoxanthin ÁSargassum siliquastrum ÁCell damage ÁOxidative stress ÁReactive oxygen speciesIntroductionReactive oxygen species (ROS),which include free radi-cals such as superoxide anion radical (O 2-),hydroxyl radicals (ÁOH)and non free-radical species such as H 2O 2and singlet oxygen (1O 2),formed during normal metabolic processes,which can easily initiate the peroxidation of membrane lipids,leading to the accumulation of lipid peroxides.Free radical scavengers and antioxidants can reduce lipid peroxidation and the generation of ROS.The importance of antioxidants in human health has become increasingly clear due to spectacular advances in under-standing the mechanisms of their reaction with oxidants.Furthermore,interest in employing antioxidants from nat-ural sources to increase the shelf-life of foods is considerably enhanced by consumer preference for natural ingredients and concerns about the toxic effects of syn-thetic antioxidants [1–3].Algae,as photosynthetic organisms,are exposed to a combination of light and high oxygen concentrations what induces the formation of free radicals and other oxidative reagents.The absence of structural damage in the algae leads to consider that these organisms are able to generate the necessary compounds to protect themselves against oxidation [4].In this respect,algae can be considered as anS.-J.Heo ÁS.-C.Ko ÁS.-M.Kang ÁK.-W.Lee ÁY.-J.Jeon Faculty of Applied Marine Science,Cheju National University,Jeju 690-756,South Koreae-mail:skysjheaven@H.-S.Kang ÁJ.-P.KimKRIBB,52Eoeun-dong,Yuseong,Daejeon 305-806,South KoreaS.-H.KimDepartment of Food Bioengineering,Cheju National University,Jeju 690-756,South KoreaM.-G.ChoFood and Biotechnology,Dongseo University,Pusan 617-716,South KoreaY.-J.Jeon (&)Marine and Environmental Research Institute,Cheju National University,Jeju 695-814,South Korea e-mail:youjinj@cheju.ac.krEur Food Res Technol (2008)228:145–151DOI 10.1007/s00217-008-0918-7important source of antioxidant compounds that could be suitable for protecting our bodies against the ROS formed e.g.by our metabolism or induced by external factors(as pollution,stress,UV radiation,etc.).Fucoxanthin is found in edible brown algae and,along with b-carotene,is one of the most abundant carotenoids found in nature[5].There have recently been several reports that fucoxanthin showed biological activities such as radical scavenging,antiproliferative,antiobesity,anti-tumor,and antiangiogenic activities[6–10].Nonetheless, the protective effects of cell damages related to antioxidant activities of fucoxanthin have not yet been reported. Accordingly,the present study isolated the marine natural carotenoid fucoxanthin from Sargassum siliquastrum and evaluated its cytoprotective effects against H2O2-induced cell damage.Materials and methodsMaterialsThe marine alga S.siliquastrum was collected along the coast of Jeju Island,Korea,between October2005and March2006.The samples were washed three times with tap water to remove the salt,epiphytes,and sand attached to the surface,then carefully rinsed with fresh water,and maintained in a medical refrigerator at-20°C.Therefore, the frozen samples were lyophilized and homogenized with a grinder prior to extraction.General experimental proceduresOptical rotations were measured on a JASCO P-1020 polarimeter.The UV and FT-IR spectra were recorded on a Pharmacia Biotech Ultrospec3000UV/Visible spec-trometer and a SHIMAZU8400s FT-IR spectrometer, respectively.NMR spectra were recorded on a Varian INOVA400MHz NMR spectrometer.CD3OD was used as a solvent for the NMR experiments,and the solvent signals were used as an internal reference.ESI and HREI mass spectra acquired using a Finnigan Navigator30086and JMS-700MSTATION high resolution mass spectrometer system,respectively.The HPLC was carried out on a Waters HPLC system equipped with a Waters996photo-diode array detector and Millenium32software using C18 column(J’sphere ODS-H80,150mm920mm,4l m, YMC Co.).Extraction and isolationThe powdered S.siliquastrum(400g)was extracted with 80%aqueous MeOH,and was evaporated under vacuo.Then,the MeOH extract was partitioned with CHCl3.The chloroform extract(9g)was fractionated by silica column chromatography using stepwise elution with CHCl3–MeOH mixture(100:1?1:1)to afford separated active fractions.A combined active fraction was further subjected to a Sephadex LH-20column saturated with100%MeOH, and thenfinally purified by reversed-phase HPLC to give fucoxanthin(74.9mg).1H and13C NMR data were assigned in Table1and the structure was illustrated in Fig.1.Cell cultureCells of a monkey kidneyfibroblast line(Vero)were maintained at37°C in an incubator with humidified atmosphere of5%CO2.Cells were cultured in Dulbecco’s modified Eagle’s medium containing10%heat-inactivated fetal calf serum,streptomycin(100l g/mL),and penicillin (100U/mL).Intracellular ROS measurementFor detection of intracellular ROS,Vero cells were seeded in96-well plates at a concentration of19105cells/mL. After16h,the cells were treated with various concentra-tions of fucoxanthin,and incubated at37°C under a humidified atmosphere.After30min,H2O2was added at a concentration of1mM,and then cells were incubated for additional30min at37°C.Finally,20,70-dichlorodihy-drofluorescein diacetate(DCFH-DA;5l g/mL)was introduced to the cells,and20,70-dichlorofluorescein(DCF)fluorescence was detected at an excitation wavelength of 485nm and an emission wavelength of535nm,using a Perkin-Elmer LS-5B spectrofluorometer.The percentage of intracellular ROS scavenging activity was calculated in accordance with the following equation:Intracellular ROS scavenging activity%ðÞ¼1ÀC1=C0ðÞ½ Â100where C1is thefluorescence intensity of cells treated with H2O2and fucoxanthin,and C0is thefluorescence intensity of cells treated with H2O2and distilled water instead of fucoxanthin.Assessment of cell viabilityCell viability was then estimated via an3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide(MTT) assay,which is a test of metabolic competence predicated upon the assessment of mitochondrial performance.It is colorimetric assay,which is dependent on the conversion of yellow tetrazolium bromide to its purple formazan derivative by mitochondrial succinate dehydrogenase inviable cells[11].The cells were seeded in96-well plate at a concentration of19105cells/mL.After16h,the cells were treated with fucoxanthin at difference concentrations. Then,10l L of H2O2(1mM)was added to the cell culture medium,and incubated for24h at37°C.MTT stock solution(50l L;2mg/mL)was then applied to the wells, to a total reaction volume of200l L.After4h of incu-bation,the plates were centrifuged for5min at8009g,and the supernatants were aspirated.The formazan crystals in each well were dissolved in150l L of dimethylsulfoxide (DMSO),and the absorbance was measured via ELISA at a wavelength of540nm.Relative cell viability was evalu-ated in accordance with the quantity of MTT converted to the insoluble formazan salt.The optical density of the formazan generated in the control cells was considered to represent100%viability.The data are expressed as mean percentages of the viable cells versus the respective control.Determination of DNA damage by comet assayComet assay was performed to determine the oxidative DNA damage[12].The cell suspension was mixed with 75l L of0.5%low melting agarose(LMA),and added to the slides precoated with1.0%normal melting agarose. After solidification of the agarose,slides were covered with another75l L of0.5%LMA and then immersed in lysis solution(2.5M NaCl,100mM EDTA,10mM Tris,andTable1NMR spectroscopic data for fucoxanthin in CDCl3Position13C1H(mult.J=Hz)Position13C1H(mult.J=Hz) 135.81035.2247.1 1.36(1H,dd,J=8.7,14.2)1.49(1H,dd,J=14.2)2045.4 1.41(1H,dd,J=10.4,14.9)2.00(1H,dd,J=2.9,14.9)364.3 3.80(1H,m)3068.0 5.37(1H,tt,J=8.8,12.0)441.6 1.77(1H,dd,J=8.7,14.2)2.29(1H,dd,J=2.9,17.8)4045.2 1.53(1H,dd,J=10.4,14.9)2.29(1H,dd,J=2.9,17.8)566.25072.7667.160117.5740.8 2.59,3.64(2H,d,J=20.4)70202.48170.580103.4 6.04(1H,s)9134.590132.510139.17.14(1H,d,J=12.8)100128.5 6.12(1H,d,J=11.6) 11123.4 6.58(1H,m)110125.7 6.71(1H,t,J=12.0) 12145.0 6.66(1H,t,J=12.8)120137.1 6.34(1H,d,J=11.6) 13135.4130138.114136.6 6.40(1H,d,J=11.6)140132.2 6.26(1H,d,J=11.6) 15129.4 6.67(1H,m)150132.5 6.71(1H,t,J=12.0,14.2) 1625.0 1.37(3H,s)16029.2 1.37(3H,s)1728.10.95(3H,s)17032.1 1.06(3H,s)1821.2 1.34(3H,s)18031.3 1.34(3H,s)1911.8 1.80(3H,s)19014.0 1.80(3H,s)2012.8 1.98(3H,s)20012.9 1.98(3H,s)30OAc,CH321.4 2.03(3H,s)30OAc,C=O197.9Data were obtained using400MHz for1H and100MHz for13C1%sodium lauryl sarcosine;1%Triton X-100and10% DMSO)for1h at4°C.The slides were next placed into an electrophoresis tank containing300mM NaOH and 10mM Na2EDTA(pH13.0)for40min for DNA unwinding.For electrophoresis of the DNA,an electric current of25V/300mA was applied for20min at4°C. The slides were washed three times with a neutralizing buffer(0.4M Tris,pH7.5)for5min at4°C,and then treated with ethanol for another5min before staining with 50l L of ethidium bromide(20l g/mL).Measurements were made by image analysis(Kinetic Imaging,Komet5.0, UK)andfluorescence microscope(LEICA DMLB,Ger-many),determining the percentage offluorescence in the tail(tail intensity;50cells from each of two replicate slides).Nuclear staining with Hoechst33342The nuclear morphology of the cells was evaluated using the cell-permeable DNA dye,Hoechst33342.Cells with homogeneously stained nuclei were considered viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis[13,14].The cells were placed in24-well plates at a concentration of 19105cells/mL.Sixteen hours after plating,the cells were treated with various concentration of fucoxanthin,and further incubated for1h prior to expose to H2O2(1mM). After24h,1.5l L of Hoechst33342(stock10mg/mL),a DNA-specificfluorescent dye,were added to each well, followed by10min of incubation at37°C.The stained cells were then observed under afluorescence microscope equipped with a CoolSNAP-Pro color digital camera,in order to examine the degree of nuclear condensation.Statistical analysisThe data are expressed as the mean±standard error.A statistical comparison was performed via the SPSS package for Windows(Version10).p-Values of\0.05were con-sidered to be significant.Results and discussionCells are protected from ROS-induced damage by a variety of endogenous ROS-scavenging enzymes,chemical com-pounds,and natural products.Recently,interest has been increased in the therapeutic potential of natural products to be used as natural antioxidants in the reduction of such freeradical-induced tissue injuries,thereby suggesting that many natural products may prove to possess therapeuti-cally useful antioxidant compounds [15–17].H 2O 2has been extensively used as an inducer of oxi-dative stress in in vitro models.It readily crosses the cellular membranes giving rise to the highly reactive hydroxyl radical,which has the ability to react with mac-romolecules,including DNA,proteins,and lipids,and ultimately damage to whole cell [18,19].Several studies have shown that oxidative stress is a major cause of cellular injuries in a variety of human diseases including cancer,neurodegenerative,and cardiovascular disorders [20–22].Recently,many researchers have made considerable efforts to search natural antioxidants.Many studies have revealed that seaweeds have potential to be used as a candidate for natural antioxidant.Potent antioxidative compounds have already been isolated from seaweeds and identified as phylopheophytin in Eisenia bicyclis (arame),fucoxanthine in Hijikia fusiformis (hijiki),phlorotannin in Ecklonia stolonifera ,bromophenols in Polysiphonia urceolata ,and sulfated polysaccharides [6,23–27].Among these antiox-idants,fucoxanthine,a kind of carotenoid,showed radical scavenging activity.In this study,we investigated the antioxidant effects of natural carotenoid fucoxanthin,after the administration of H 2O 2in cell lines.20,70-Dichlorodihydrofluorescein diacetate was used as a probe for ROS measurement.DCFH-DA crosses cell membranes and is hydrolyzed enzymatically by intracel-lular esterase to nonfluorescent DCFH.In the presence of ROS,DCFH is oxidized to highly fluorescent DCF.It is well known that H 2O 2is the principal ROS responsible for the oxidation of DCFH to DCF [28].In the presentstudy,Fig.5Photomicrographs of DNA damage and migration observed under fucoxanthin isolated from S.siliquastrum where the tail moments were decreased.a control,b cells treated with 50l M H 2O 2,c cells treated with 5l M compound +50l M H 2O 2,d cells treated with 50l M compound +50l M H 2O 2,e cells treated with 200l M compound +50l M H 2O 2we investigated scavenging activity of fucoxanthin on intracellular ROS and the results are illustrated in Fig.2.Fluorescence intensity of control cells was recorded as 3398while H 2O 2treated cells showed 7452.However,the addition of fucoxanthin to the cells mixed with H 2O 2reduced intracellular ROS accumulation to 6079,4867,and 3149at 5,50,and 100l M,respectively.Moreover,the scavenging activity of fucoxanthin on intracellular ROS was dose-dependently increased as 18.4,34.7,and 57.7%at 5,50,and 100l M,respectively.Hence these results indicate that fucoxanthin has efficient antioxidant proper-ties which can be developed into a potential bio-molecular candidate to inhibit ROS formation of cellular.To evaluate whether fucoxanthin protect from cellular damage induced by H 2O 2,cells were pretreated with fuco-xanthin for 24h in the absence or presence of oxidative stress.After then,cell viability was measured via MTT assay.As shown in Fig.3,H 2O 2treatment without fuco-xanthin decreased cell viability to 43.7%,while fucoxanthin prevented cells from H 2O 2-induced damage,restoring cell survival to 63.6,69.4,78.5,and 89.2%at the concentrations of 5,50,100,and 200l M,respectively.Active mitochon-dria of living cells can cleave MTT to produce formazan,the amount of which is directly correlated to the living cell number [29].The results of MTT assay showed that for-mazan content was reduced due to H 2O 2treatment,however,significantly increased with addition of fucoxan-thin.These results suggest that fucoxanthin has ability to protect vero cells from oxidative stress-related cellular injuries.The protective effect of fucoxanthin on DNA damages was also confirmed by comet assay and was presented as tail DNA percent.This assay can reflect different types of DNA damage,such as DNA single strand breakage,or incomplete DNA repairing and shows high sensitivity in detecting oxidative stress [30].As shown in Fig.4,H 2O 2treatment increased the tail moments in the cells versus the control cells.However,the increased tail moments caused by H 2O 2induced oxidative DNA damage were decreased in the cells treated with fucoxanthin.The inhibitioneffectFig.6Protective effect of fucoxanthin isolated from S.siliquastrum against H 2O 2-induced apoptosis in vero cells.Cellular morphological changes were observed under afluorescence microscope after Hoechst 33342staining.a untreated,b 500l M H 2O 2,c 5l M compound +500l M H 2O 2,d 50l Mcompound +500l M H 2O 2,e 200l Mcompound +500l M H 2O 2.Apoptotic bodies are indicated by arrowsof fucoxanthin on DNA damage was38.8,52.5,and63.4% at the concentrations of5,50,and200l M,respectively. The DNA migration profiles of tested cells when treated with different concentrations of fucoxanthin are presented in Fig.5.The DNA was completely damaged and the amount of tail DNA was significantly increased in the group treated with only H2O2(Fig.5b),compared to the control(Fig.5a).However,the amount of tail DNA was increasingly decreased with increased concentrations of fucoxanthin(Fig.5c–e).In a previous study,Kang et al.[23]reported that some natural compounds from brown seaweeds have an ability to increase catalase located at peroxisome in cell that converts H2O2into molecular oxygen and water.In this study,we showed that fuco-xanthin has prominent effect on the H2O2-induced DNA damage.The inhibition activities on the DNA damage and the decrease of the amounts of tail DNA might be related to the ability of fucoxanthin to increase catalase.The protective effect of fucoxanthin on apoptosis induced by H2O2was investigated by nuclear staining with Hoechst33342.Hoechst33342dye specifically stains DNA,and is widely used to detect the nuclear shrinkage such as chromatin condensation,nuclear fragmentation, and the appearance of apoptotic bodies,which are all indications of apoptosis[31].The microscopic photograph in Fig.6,the negative control,which contained no com-pound or H2O2,possessed intact nuclei(Fig.6a),and the positive control(H2O2treated cells)exhibited significant nuclear fragmentation,indicating apoptosis(Fig.6b). However,the addition of fucoxanthin with H2O2reduced the apoptotic bodies(Fig.6c–e),which suggested the ability of fucoxanthin to protect against nuclear fragmen-tation amidst oxidative stress.In conclusion,the results obtained in the present study show that fucoxanthin isolated from S.siliquastrum can effectively inhibit intracellular ROS formation,DNA damage,and apoptosis induced by H2O2.Moreover, fucoxanthin showed evidence in cell survival rate.These results revealed that fucoxanthin can be used not only as the easily accessible source of natural antioxidants but also as an ingredient of the functional food and cosmetic agents related to the prevention and control oxidative stress. Acknowledgements This research was supported by a grant(M-2007-03)from the Marine Bioprocess Research Center of the Marine Bio21Center,funded by Ministry of Marine Affairs and Fisheries, Republic of Korea.References1.Schwarz K,Bertelsen G,Nissen LR,Gardner PT,Heinonen MI,Hopia A,Huynh-Ba T,Lambelet P,McPhail D,Skibsted LH, Tijburg L(2001)Eur Food Res Technol212:319–3282.Farag RS,El-Baroty GS,Basuny AM(2003)Int J Food SciTechnol38:81–873.Heo SJ,Park EJ,Lee KW,Jeon YJ(2005)Bioresour Technol96:1613–16234.Jimenez-Escrig A,Jimenez-Jimenez I,Pulido R,Saura-Calixto F(2001)J Sci Food Agric81:530–5345.Berhard K,Moss GP,Toth GY,Weedon BLC(1976)Tetrahe-dron Lett17:115–1186.Yan X,Chuda Y,Suzuki M,Nagata T(1999)Biosci BiotechnolBiochem63:605–6077.Hosokawa M,Kudo M,Maeda H,Kohno H,Tanaka T,MiyashitaK(2004)Biochim Biophys Acta1675:113–1198.Maeda H,Hosokawa M,Sashima T,Funayama K,Miyashita K(2005)Biochem Biophys Res Commun332:392–3979.Kotake-Nara E,Asai A,Nagao A(2005)Cancer Lett220:75–8410.Sugawara T,Matsubara K,Akagi R,Mori M,Hirata T(2006)JAgric Food Chem54:9805–981011.Mosmann T(1983)J Immunol Methods65:55–6312.Singh NP(2000)Mutat Res455:111–12713.Gschwind M,Huber G(1995)J Neurochem65:292–30014.Lizard G,Fournel S,Genestier L,Dhedin N,Chaput C,FlacherM,Mutin M,Panaye G,Revillard JP(1995)Cytometry21:275–28315.Lee SE,Ju EM,Kim JH(2002)Exp Mol Med34:100–10616.Jung WK,Rajapakse N,Kim SK(2005)Eur Food Res Technol220:535–53917.Ahn GN,Kim KN,Cha SH,Song CB,Lee J,Heo MS,Yeo IK,Lee NH,Jee YH,Kim JS,Heo MS,Jeon YJ(2007)Eur Food Res Technol226:71–7918.Satoh T,Sakai N,Enokido Y,Uchiyama Y,Hatanaka H(1996)JBiochem120:540–54619.Halliwell B,Clement MV,Long LH(2000)FEBS Lett486:10–1320.Al-Enezi KS,Alkhalaf M,Benov LT(2006)Free Radic Biol Med40:1144–115121.Zhang L,Yu H,Sun Y,Lin X,Chen B,Tan C,Cao G,Wang Z(2007)Eur J Pharmacol564:18–2522.Strazzullo P,Puig JG(2007)Nutr Metab Cardiovasc Dis17:409–41423.Kang HS,Chung HY,Jung HA,Son BW,Choi JS(2003)ChemPharm Bull51:1012–101424.Cahyana AH,Shuto Y,Kinoshita Y(1992)Biosci BiotechnolBiochem56:1533–153525.Li K,Li XM,Ji NY,Wang BG(2007)Bioorg Med Chem15:6627–663126.Zhang Q,Li N,Liu W,Zhao Z,Li Z,Xu Z(2004)Carbohydr Res339:105–11127.Kim SH,Choi DS,Athucorala Y,Jeon YJ,Senevirathne M,RhaCK(2007)Int J Food Sci Nutr12:65–7328.LeBel CP,Ischiopoulos H,Bondy SC(1992)Chem Res Toxicol5:227–23129.Hu Z,Guan W,Wang W,Huang L,Xing H,Zhu Z(2007)CellBiol Int31:798–80430.Senthilmohan ST,Zhang J,Stanley RA(2003)Nutr Res23:1199–121031.Kerr JF,Gobe GC,Winterford CM,Harmon BV(1995)MethodsCell Biol46:1–27。
中英文造纸专业词汇
Gu3ha ng2dia n4do ng4ji1带有)鼓行电动机
Troubleshoot ing
Gu4zha ng4pai2chu2故障排除
Channel doctor
Gua1dao1cao2刮刀槽
Doctor blade
Gua1dao1pia n4刮刀片
Doctor
Gua1dao1刮刀
Bia n4j in g4变径
Shifting fork
Bia n4su4cha1变速叉
Surface
Biao3mia n4表面
Peel off (sollevare, lift)
Bo1li2 (vetroe bo1li)
Lift-off hose
Bo1li2rua n3gua n3剥离软管
Glass fiber
Dao3gu n3导辊
Lead-i n roll
Dao3ru4gu n3导入辊
Collapse
Dao3ta1倒塌
Plasma device
Den g3li2zi qielgeliil等离子切割机
Baseme nt (floor)
dilcha n3低产
Zone
Di4dai4地带
An chor bolt
Scraper
Gua1shui3ba n3刮水板
Pipe
Gua n3dao4管道
Pipi ng
gua n3lu4管路
Grout ing
gua n4jia ng1灌浆
Tank
Guan4zi
Finish (fin itura)
Gua ng1jie2du4光洁度
Fiber optics
Gua ng1xia n1光纤
(完整版)基于CHO细胞的单抗生产2
细胞库的相关法规要求
细胞库的检定
细胞库检定-CHO细胞
检测项目
细胞鉴别 细菌、真菌检查
MCP
+ +
WCP
+ +
分歧杆菌检查
+
+
支原体检查
(+)
(+)
细胞形态观察及血吸附试验
+
+
内 体外不同细胞接种培养法
+
+
、 动物和鸡胚体内接种法
+
-
源 病
逆转录病毒检查
+
-
毒 种属特异性病毒检查（鼠源）
(+)
荧光强度值能反映细胞
目标的相对表达水平
筛选方法
➢ ClonePix
Semi solid media
筛选方法
➢ Limiting dilution
有限稀释法(limiting dilution cloning, LDC)是一种通过梯度稀释以获得单 克隆的方法。 它的应用范围广, 对于大多数细胞类型, 如杂交瘤细胞、 CHO细胞及干细胞等都有较好的克隆分离效果。 LDC的操作过程大致 分为接种(铺板)、 筛选和扩增。 接种的细胞液经过逐级稀释后加到96 孔板中, 每个孔所含的细胞个数理论上不大于1
载体的一般性构成
1、IRES：即内部核糖体进入位点，是一段核酸序列，它的存在能够使蛋白质翻
重组人促红素β注射液(CHO细胞)
重组人促红素β注射液(CHO细胞)【药品名称】通用名称：重组人促红素β注射液(CHO细胞)英文名称：Recombinant Human Erythropoietin β Injection (CHO Cell)【成份】主要组成成份:重组人红细胞生成素-β辅料包括：尿素，氯化钠，聚山梨醇酯-20，二水合磷酸二氢钠，十二水合磷酸氢二钠，二水合氯化钙，甘氨酸，左旋亮氨酸，左旋异亮氨酸，左旋苏氨酸，左旋谷氨酸，左旋苯丙氨酸，注射用水。
【适应症】本品适用于因慢性肾衰竭引致贫血，包括行血液透析、腹膜透析和非透析治疗者。
治疗接受化疗的非髓性恶性肿瘤成人患者的症状性贫血。
本品用于治疗贫血时，仅在出现贫血症状时方可使用。
【用法用量】应由在上述适应症领域中有丰富经验的医生指导下进行罗可曼治疗。
由于在个别的病例中观察到过敏样反应，建议在医学监护下进行首次给药。
应注意本品只有在溶液澄清或呈轻微乳状，无色且几乎无可见颗粒时方可用于注射。
欲重拾注射器中的药品是无菌的，但未作防腐处理。
在任何情况下，每支注射器都不可以多次注射。
治疗成人和儿童慢性肾功能衰竭患者的症状性贫血。
患者贫血的症状和后遗症随着年龄，性别和疾病总负荷的不同而有所不同；医生有必要对个体患者的临床病程和状态进行评价。
可以通过皮下或静脉注射罗可曼来升高血红蛋白的水平，使其不超过12g/dl(7.5mmol/l)。
对于未进行血液透析的患者，首选皮下注射给药以避免刺穿外周静脉。
如果静脉注射给药，应在约2分钟内完成，例如，对于血液透析患者在透析结束时经动静脉瘘管注入。
因为病人的个体差异，临床上可以观察到个别患者的血红蛋白值偶尔超过或低于预期的血红蛋白水平。
考虑到血红蛋白靶浓度在10g/dl （6.2mmol/l）到12g/dl（7.5mmol/l）之间，可以通过剂量管理达到不同的血红蛋白水平。
应该避免出现持续的血红蛋白水平超过12g/dl(7.5mmol/l)；当血红蛋白值超过12g/dl(7.5mmol/l)时，可根据以下指导原则调整剂量：应当避免出现用药4周后血红蛋白水平升高幅度超过2g/dl(1.25mmol/l)。
无血清培养CHO细胞的研究进展
Science &Technology Vision 科技视界随着生物技术的飞速发展,单克隆抗体、细胞因子、疫苗和各种生物活性蛋白等生物制品在诊断和治疗中应用日益广泛,其需求量大大增加,因此,通过体外大规模培养动物细胞生产生物制品技术的研究越来越受到青睐。
CHO 细胞是生产蛋白类生物制品最重要的表达系统,采用无血清培养CHO 细胞相比含血清培养,能避免对实验动物的伤害、潜在的污染源影响、不同批次血清间的生物活性和因子的不一致及价格高等诸多弊端,因而受到生物制药领域的广泛关注。
1CHO 细胞CHO 细胞即中国仓鼠卵巢细胞(Chinese Hamster Ovary Cell),是最具代表性的动物细胞表达系统,被广泛应用于生物制药领域。
它建株于1957年,由美国科罗拉多大学Theodore T.Puck 从一成年雌性中国仓鼠卵巢中分离得到,是一种连续细胞系,能传百代以上,具有永生性。
后来陆续又有很多科学家用药物加压、极端条件筛选和诱导突变等方法筛选得到了一系列不同表型的CHO 细胞株[1],如DUKX-B11、DG-44和CHO-K1等。
CHO 细胞能用于表达各种复杂的重组蛋白。
据统计,70%以上的动物细胞表达药物产品都是以CHO 细胞为表达宿主[2]。
2011年6月批准的27种单克隆抗体中,其中13种就是通过CHO 细胞表达的,占了约50%的比例。
CHO 细胞之所以成为最受欢迎的宿主细胞,主要在于其易于培养以及基因操作,而且相比其他表达系统,它还具有许多优点[3-4]:①具有准确的转录后修饰功能,表达的糖基化药物蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分子;②具有产物胞外分泌功能,便于下游产物分离纯化;③具有外源重组基因的高效扩增和表达能力;④既可贴壁生长也可以进行悬浮培养,且有较高的耐受剪切力和渗透压能力,外源蛋白表达水平较高;⑤CHO 细胞属于成纤维细胞,很少分泌自身的内源蛋白,利于外源蛋白的后分离。
康天晟合生物技术有限公司QuaCell
Version 3.0Revision date: Aug.09,2019产品说明书简介QuaCell ®CHO CD02培养基可为中国仓鼠卵巢细胞(CHO 细胞)的高密度增殖提供丰富的营养成分。
此产品是一款无血清、无动物成分、化学限定培养基，适用于悬浮培养CHO 细胞以表达抗体及蛋白产物。
只需简单的驯化甚至不用驯化，就可从含血清培养基或其他无血清培养基中，直接传代接种至QuaCell ® CHO CD02培养基中。
此培养基配方中不含次黄嘌呤、胸苷及L-谷氨酰胺，适合二氢叶酸还原酶、谷氨酰胺合成酶（GS）筛选系统。
组分L-谷氨酰胺 不含，用前按需添加 葡萄糖 6.0 g/L 次黄嘌呤&胸苷 不含 酚红 不含 碳酸氢钠 1.90g/L 水解产物不含产品用途在处理或补充培养基时使用无菌技术。
本产品用于研究或进一步制造使用。
警告：不用于人类或动物治疗用途。
超出规定范围的使用可能会触犯当地法律。
安全信息阅读物料安全数据表（MSDS）并依据相关的安全操作规范，佩戴适当的护目镜，洁净服，口罩和手套等。
用前准备• QuaCell ®CHO CD02培养基的使用需要无菌• 产品不含L-谷氨酰胺；用于正常表达GS 基因的宿主细胞的增殖培养时，建议在使用前添加4mM L-谷氨酰胺；根据细胞株GS 基因编辑情况，可按需调节L-谷氨酰胺浓度。
• 不推荐使用抗生素。
• 开封后未用完的培养基应进行分装，使用封口膜封口，在2～8℃避光保存。
产品外包装副标提供一组印有产品信息的不干胶便签，可撕下使用，便于分装标记及实验记录。
培养条件培养基：完全的QuaCell ®CHO CD02 细胞系：CHO cells 培养类型：悬浮培养容器：摇瓶/TPP/反应器 温度范围：37℃±0.5培养箱气体要求：5～8% CO 2的加湿培养注意：确保适当的气体交换和最小化的曝光培养。
细胞复苏1. 在37℃水中快速解冻（<2分钟）冻存管中的细胞液；2. 将细胞液转移至15 ml 离心管中，加入10ml 预热的QuaCell ®CHO CD02培养基，1000rpm 离心3分钟，丢弃上清液，使用5 ml QuaCell ®CHO CD02培养基重悬，计数；3. 将冷冻管的全部细胞液转移到装有15 ml 预热的完全QuaCell ®CHO CD02培养基的125 ml 摇瓶中，稀释至所需细胞密度；4. 在含有5～8% CO 2，37℃，加湿的培养箱或摇床进行培养，培养时拧松瓶盖或使用通气盖以进行气体交换； 5. 细胞复苏后培养2～5天处于对数生长中期时传代。
基于CHO细胞的单抗生产PPT
单克隆拍照系统
FDA目前对于单克隆证明方法意见
克隆性
FISH不但可用于已知基因或序列的染色体定位，而且也可用于未克隆基因或遗传 标记及染色体畸变的研究。在基因定性、定量、整合、表达等方面的研究中颇 具优势。
筛选检定
细胞代次的规定
细胞库的相关法规要求
细胞库的相关法规要求
➢ ClonePix /Cell Xpress TM 克隆筛选及细胞表达系统
➢ Limiting dilution 有限稀释法
筛选方法
➢ Fluorescence-Activated Cell Sorting (FACS)-Based Screening
The cold capture method used in FACS where fluorescently labeled antibodies bind to secreted
载体的一般性构成
1、IRES：即内部核糖体进入位点，是一段核酸序列，它的存在能够使蛋白质翻
译起始不依赖于5‘帽结构，从而使直接从信使RNA（mRNA）中间起始翻译成 为可能。通常来讲，真核生物翻译只能从mRNA的5‘端开始，因为翻译起始必 须依赖于5’端的帽子结构。
2、Promoters：即启动子，控制基因表达（转录）的起始时间和表达的程度，启 动子本身并不控制基因活动，而是通过与称为转录因子（transcription factor）的这种蛋白质（proteins）结合而控制基因活动的 抗体序列需使用强启动子如SV40启动子等 而抗性基因需要使用削弱的启动子
基于CHO细胞的单抗生产（第一版）
汪洋 2018/02
目录
• CHO细胞的构建 • CHO细胞的表达 • CHO细胞表达后的产物纯化
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CHO-敲除细胞系简介：CHO细胞又称中国仓鼠卵巢细胞，是一种来源于中国仓鼠卵巢的上皮细胞系。
中国仓鼠卵巢（CHO）细胞以其独特的特性，已成为制备治疗性重组蛋白的主要工具。
这种细胞系通常用于生物学和医学研究。
同时，它也是最广泛使用的哺乳动物宿主在工业生产治疗蛋白方面。
应用程序：基因打靶法构建基因敲除CHO细胞系由于CHO细胞系是治疗性抗体产生的主要宿主，构建高效的CHO细胞系具有重要意义。
在构建CHO细胞系中，常用的转染方法有两种，一种是随机整合法，另一种是基因靶向法。
前者导致抗体产生率的变化，通常会影响转基因表达水平。
然而，在基因打靶方法中，外源基因被插入特定的染色体区域。
这种方法是基于利用针对宿主细胞特定基因组区域的序列进行同源重组。
研究人员利用CRISPR/Cas9系统作为基因靶向方法，通过引导RNA和Cas9诱导双链断裂，提高同源重组效率。
因此，CRISPR/Cas9载体系统可以有效地将外源基因插入CHO细胞系中。
利用谷氨酰胺合成酶基因敲除细胞提高CHO细胞系的产生效率尽管中国仓鼠卵巢（CHO）细胞以其独特的特性，已经成为制造治疗性重组蛋白的主要工具，但如何有效地鉴定这些稀有的CHO细胞系是CHO细胞系产生的主要挑战之一，在大量低产和低产无性系中的高产无性系。
目前，有两种主要的CHO表达系统被广泛应用，基于二氢叶酸还原酶（DHFR）的甲氨蝶呤（MTX）选择和基于谷氨酰胺合成酶（GS）的甲硫氨酸硫氧胺（MSX）选择。
为了研究内源性GS表达对选择效率的潜在影响，采用锌指核酸酶（ZFN）技术，以内源性CHO-GS基因为靶点，构建了GS基因敲除CHOK1SV细胞株。
双等位基因修饰CHO-GS基因的高效性（∼2%）支持了ZFN技术的独特优势，特别是在CHO 细胞中。
通过对所有GS基因敲除细胞系谷氨酰胺依赖性生长的观察，证实了GS 酶功能的破坏。
谷氨酰胺合成酶介导的CHO细胞单克隆抗体筛选利用GS基因敲除CHO细胞系12和启动子工程，即使在没有MSX1的情况下，基于GS的系统也能有效地产生细胞系。
利用两个不同的宿主细胞系（CHO-K1和GS-knockout-CHO，GS-KO），在不同MSX浓度下，通过单轮筛选产生mAb的rCHO细胞克隆。
GS基因敲除CHO细胞系具有改进的选择严格性。
使用GS基因敲除CHO宿主细胞系有助于在没有MSX的情况下快速产生高产克隆，降低乳酸和氨的产量。
mAb克隆产生和长期培养的生产稳定性试验流程示意图。
CRISPR/Cas9基因敲除HEK293细胞系的策略敲除HEK293细胞株的工作流程参考文献:Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC, Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.Aga, M., Yamano, N., Kumamoto, T. et al. Construction of a gene knockout CHO cell line using a simple gene targeting method. BMC Proc 9, P2 (2015).Noh, S.M., Shin, S. & Lee, G.M. Comprehensive characterization of glutaminesynthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8, 5361 (2018).CHO ko cell lineIntroduction:CHO cells are also called Chinese hamster ovary cells, which are an epithelial cell line that originated from the ovary of the Chinese hamster. Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins. This cell line is usually used in biological and medical research. Also, it is the most widely used mammalian host in terms of the industrial production of therapeutic proteins.Application:Construction of a gene knockout CHO cell line using gene targeting methodSince CHO cell line is a major host for therapeutic antibody production, constructing productive CHO cell lines is important. There are two major transfection methods that are commonly used in building characterized CHO cell lines, one is random integration and the other one is gene targeting. The former one, which causes variation in antibody productivity, would usually affect transgene expression levels. However, in gene-targeting methods, exogenous genes are inserted into a specific chromosomal region. This method is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Researchers used CRISPR/Cas9 system as a gene-targeting method, which induces double-strand breaks(DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination. Therefore, CRISPR/Cas9 vector system can efficiently insert exogenous genes into CHO cell lines.Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cellsAlthough Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. Currently, there are two main CHO expression systems that have been widely used, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection.To study endogenous GS expression's potential impact on selection efficiency,GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of the glutamine-dependent growth of all GS-knockout cell lines.Characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodiesWith a GS-knockout CHO cell line12 and promoter engineering, the GS-based system can be effective in cell line generation even in the absence of MSX1. Using two different host cell lines (CHO-K1 and GS-knockout CHO (GS KO), mAb producing rCHO cell clones were generated by a single round of selection at various MSX concentrations.GS-knockout CHO cell lines with an improved selection stringency. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.A schematic diagram of the process for mAb producing clone generation andlong-term culture for testing the production stability.A schematic diagram of the process for mAb producing clone generation and long-term culture for testing the production stability.CRISPR/Cas9 knockout HEK293cell line strategiesWork flow of knockout HEK293cell lineReference:Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC, Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.Aga, M., Yamano, N., Kumamoto, T. et al. Construction of a gene knockout CHO cell line using a simple gene targeting method. BMC Proc 9, P2 (2015).Noh, S.M., Shin, S. & Lee, G.M. Comprehensive characterization of glutaminesynthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8, 5361 (2018).。