Evaluation of Chinese hamster ovary cell stability during repeated batch culture for large-scale
Evaluation of Chinese hamster ovary cell stability during repeated batch culture for
large-scale antibody production
Yoshihiro Kaneko,1,2,?Ryuji Sato,3and Hideki Aoyagi 2
Bio-product Technology Research Department,Drug Engineering Division,Chugai Pharmaceutical Co.,Ltd.,5-1,Ukima 5-Chome,Kita-ku,Tokyo 115-8543,Japan 1Life Science and Bioengineering,Graduate School of Life and Environmental Sciences,University of Tsukuba,Tsukuba,Ibaraki 305-8572,Japan 2and Utsunomiya Plant Manufacturing Section 3,Chugai Pharmaceutical Manufacturing Co.,Ltd.,16-3,Kiyohara Kogyo Danchi,
Utsunomiya,Tochigi 321-3231,Japan 3
Received 17July 2009;accepted 15September 2009
Available online 14October 2009
Pharmaceutical manufacturing plants can be operated continuously for several months.It is therefore important to use cells with long-term stability for the production of active ingredients.We investigated the reliability and long-term stability of an antibody-producing cell line.A recombinant Chinese hamster ovary (CHO)cell line was cultivated in spinner flasks and reactors,including a practical production-scale reactor (1600L),for 109days to produce monoclonal antibodies against the HM1.24antigen.During cultivation,the cells remained stable and there was an increase in the rate of cell proliferation,yielding viable cells at high density.A decrease in cell-specific productivity was associated with this increase in the rate of cell proliferation.The cells were genetically stable and other measures of cellular function remained consistent throughout the cultivation period.
?2009,The Society for Biotechnology,Japan.All rights reserved.
[Key words:Recombinant CHO cell;Monoclonal antibody;HM1.24antigen;Scaled-down cell culture]
Recombinant proteins are synthesized by large-scale cultivation of genetically engineered host cells,which harbor the genes encoding the target proteins (1,2).Demand for production of recombinant proteins for biopharmaceuticals has been increasing rapidly.Produc-tion of recombinant proteins by pharmaceutical companies will serve a global market worth USD $70billion by 2010(3,4).The increasing demand for recombinant therapeutic proteins has placed significant pressure on the biopharmaceutical industry to develop high-yielding mammalian cell-based production systems.Mammalian cells are the most appropriate host cells for the production of biopharmaceuticals if complex protein structures such as monoclonal antibodies must be synthesized in their native form.Current efforts to increase the supply of recombinant protein from mammalian host cells have been directed toward increasing the scale of production rather than increasing the stability of systems for long-term production (5,6).However,increasing long-term system stability is advantageous because it saves the time normally used to produce seed cultures for large-scale bioreactors.The growth of mammalian cells is slower than that of microorganisms.The process of large-scale cultivation of mammalian cells usually involves thawing the frozen seed cells,activating cells in spinner flasks,cultivating cells in bioreactors of increasing sizes and finally cultivating cells in the production-scale bioreactor.In other words,the majority of the time spent for one batch is used to produce the seed cells used for inoculation of the main
culture.A 1000L production scale bioreactor requires at least 100L of seed cells and it usually takes more than two months to produce seed cells (cell thawing,cultivation in various sizes of spinner flasks and various sizes of bioreactors up to 100L)for inoculation and subsequent production by batch or fed-batch cell culture at scales above 1000L.These numbers imply that using the lot-to-lot batch cultivation method,only five batches can be produced from five frozen vials with the same sets of bioreactors in a year.Therefore,establishing a repeated batch culture system that would operate for much longer would enable the same practical production-scale reactor to produce,say,five lots of batch culture from only one vial of frozen cells in 3months.This saves both time and money (in terms of the number of frozen vials)and ensures product consistency.In a practical production plant,repeated batch culture is very important because it would save both the time and money required to prepare fresh seed cultures.As the frozen vials are generally prepared as pre-assessed and guaranteed master cell banks,they are extremely expensive.Thus it is very important to reduce the number of frozen vials used.
Chinese hamster ovary (CHO)cells remain stable during cultiva-tion,with relatively rapid cell proliferation that yields viable cells at high density.However,there are no published reports,assessing the stability of antibody production using CHO cell lines in large-scale (i.e.,practical-level)culture.Barnes et al.reviewed published information on the stability of protein production in hybridoma,CHO,and NS0cell lines (7).According to Barnes et al.,several reports of the instability of protein production from recombinant cell lines exist but the cause of this instability is
unknown.
Journal of Bioscience and Bioengineering
VOL.109No.3,274–280,
2010
https://www.wendangku.net/doc/60438080.html,/locate/jbiosc
?Corresponding author.Tel.:+81339688534;fax:+81339682038.E-mail address:kanekanekoysh@chugai-pharm.co.jp (Y.Kaneko).
1389-1723/$-see front matter ?2009,The Society for Biotechnology,Japan.All rights reserved.doi:10.1016/j.jbiosc.2009.09.044
In this study,as a basis for establishing a practical production system by repeated batch culture to produce seed cells continuously,we investigated long-term cell stability by assessing some key culture characteristics,such as cell-specific productivity,cell proliferation,and genetic stability.
MATERIALS AND METHODS
Cell lines Recombinant CHO (r-CHO)cells producing human monoclonal antibodies against the HM1.24antigen (mAb)were used.The HM1.24antigen is a type-II transmembrane protein that is preferentially expressed on terminally differentiated B cells and overexpressed on multiple myeloma (MM)cells (8–12).The r-CHO cells were established by transfection with a vector containing dihydrofolate reductase (dhfr)and mAb heavy-and light-chain genes into dhfr-deficient CHO cells (line DG44),followed by DHFR/methotrexate (MTX)-mediated gene amplification.A stable r-CHO cell line producing mAb was selected using 0.5μmol/L MTX,and single-cell cloning was performed by means of limiting dilution.
Cell culture media Subculturing was initiated with a spinning flask and the volume was gradually increased to fill a 160-L bioreactor.Basal media used in the subcultures were obtained by mixing CHO-S-SFM II (Gibco-Invitrogen,Carlsbad,CA,USA)and CD CHO (Gibco-Invitrogen)media.MTX,and other additives were also added to the basal medium.The concentrations of the additives were varied to obtain different concentrations of MTX and other additives.The initial glucose concentration was 4.6g/L;the medium osmotic pressure was maintained at 300±30mOsm/kg and the pH at 7.2±0.2.
The medium used for the 1600-L manufacturing-scale bioreactor (model SUS-316L,Genetic Institute,Cambridge,MA,USA)was the same as the subculture medium except that MTX was not added.The initial glucose concentration was increased to
10.0g/L;the medium osmotic pressure was maintained at 350mOsm/kg and the pH was 7.2.
Cell culture Seed and main culture Exponentially growing cells were used as an inoculum for production of the seed culture at 37°C.The scale was gradually increased from a 100ml spinner flask through a 500ml spinner flask to a 15-L bioreactor and finally to a 160-L bioreactor.The initial cell culture period for cell activation after thawing is usually longer than the subsequent culture periods during the process scale up and generally lasts for 15–30days.The condition examined in this study is the condition usually employed for actual (practical)cultivation of this cell line.The inoculation cells were maintained by repeated batch culture in the 160-L bioreactor.Cells from the 160-L bioreactor were used to inoculate the 1600-L bioreactor for the main culture.It has been reported that the expansion of the cells in the large-scale cell culture can be controlled only by changing the initial cell density (13).The pH control began on the third day of culture when the pH had decreased to 6.9.The temperature was controlled at 37°C,and the agitation speed was 40rpm.Reactivation of cells After cultivation of cells in the 1600-L bioreactor,the cells were reactivated by subculturing several times in 100ml spinner flask.The medium used was the same as that used for subcultures except that MTX was not added.Subculturing was done by transfer of exponentially growing cells at 37°C.
Scaled-down cell culture The culture was scaled down from a 160-L bioreactor to a 100-ml spinner flask for the assessment of long-term stability.We believed that reliable data on cell stability can be obtained in small scale bioreactors equipped with control devices.The cells were further subcultured by 17passages in the 100ml spinner flask.The medium used was the subculture medium with MTX.
Measurement of mAb concentration The mAb concentration in the cell culture was assayed by Protein G high performance liquid chromatography (Alliance HPLC System,Waters,Milford,MA,USA)under the following conditions:150mM NaCl/10mM phosphate buffer (pH 8.5±0.2)was used for Mobile Phase A;150mM NaCl/12mM HCl/1%acetic acid (pH 1.8±0.2)was used for Mobile Phase B;and 0.02%Na 3N/10mM phosphate buffer (pH 7.4±0.2)was used for column storage.Polysorbate 80(0.05%)/150mM NaCl/10mM phosphate buffer was used as the dilution buffer.The injection volume was 200μl /injection.A reference standard mAb was diluted 2,6,or 10times with dilution buffer to determine protein concentration.The fractions were diluted with dilution buffer to give a mAb concentration of approximately 0.1mg/ml.Elution of protein was monitored by absorbance at 280nm and the column temperature was maintained at 4°C.
Measurement of cell concentration and cell viability Trypan Blue Stain (Gibco-Invitrogen)and an erythrocytometer were used to measure cell concentration and cell viability.
Calculation of cell doubling time and cell-specific productivity The cell doubling time and cell-specific productivity were calculated from the initial and final viable cell densities and the mAb concentrations.
Measurement of glucose and lactate concentrations Glucose and lactate concentrations in culture broth were analyzed using a Biochemistry Analyzer (model 2700,Yellow Springs Instrument,Yellow Springs,OH,USA).
MTX tolerance test A lack of dhfr (dhfr ?)causes cells to become glycine,hypoxanthine,and thymidine auxotrophic.When dhfr cDNA was transformed (dhfr+)into CHO cells,only cells with the Pro-phenotype and acquired MTX tolerance were obtained.Preliminary experiments showed that the pre-production cells possessed these properties.After recovering viable cells at the end of production,cells were tested for possession of the same qualities as pre-production cells.Upon completion of
the
FIG.1.Preparation histories of EPC1,EPC2,and LEC.EPC1:cells recovered after first lot of production culture in 1600-L bioreactor;EPC2:cells recovered after second lot of production culture in 1600-L bioreactor;LEC:late-expansion cells from expansion cell culture.
TABLE 1.Cell culture profile in expansion culture.
Culture date Days Scale (L) D.T.a (h)Viability (%)
C.S.P.b (pg cell ?1day ?1)
Days 1–430.132.289.945Days 4–840.134.197.638.4Days 8–1130.122.396.038.5Days 11–1540.122.196.938.7Days 15–1830.136.498.641.8Days 18–2240.125.196.441.1Days 22–2530.127.397.539.2Days 25–2940.128.495.941.9Days 29–3230.519.598.737.8Days 29–3230.520.098.538.2Days 32–3641524.598.728.6Days 36–39316022.198.231.4Days 39–43416033.990.435.7Days 43–46316028.495.038.5Days 46–50416030.593.334Days 50–53316029.094.632.9Total average ––27.296.037.6SD c
–
–
5.3
2.8
4.2
a Doubling time.
b Cell-specifi
c productivity.c
Standard deviation.
LONG-TERM CHO CELL STABLITY DURING LARGE-SCALE CULTIVATION 275
V OL .109,2010
mAb production culture,cells were collected aseptically and inspected as follows.For testing MTX tolerance,cells were resuspended in media with or without MTX to give initial cell concentrations of 1.0to 2.0×105cells/ml.They were then incubated at 37°C
in 100ml spinner flasks for 3days.Cell growth rate,cell viability,and mAb production were compared between the two cultures.
L-Proline auxotrophy test Auxotrophy tests were performed by suspending the end-of-production cells in media with or without L-proline.The initial cell concentrations were 1.0to 2.0×105cells/ml.The cells were incubated in 100ml spinner flasks for 3days.Cell growth rate and cell viability of the cultures were compared.
Genetic stability test Southern blot and dot blot analyses were used to assess the genetic stability of the cells.
Preparation of DNA from cells Cells were washed with phosphate-buffered saline twice and re-suspended in lysis buffer (10mM Tris –HCl [pH 8.0],150mM NaCl,10mM EDTA).RNase A and sodium dodecyl sulfate (SDS)were added and the cell lysate was incubated for 30min at 37°C.Proteinase K was added to the lysate and the mixture was incubated at 55°C for 90min.Tris-saturated phenol was added and mixed (by shaking)for 20min at room temperature,and the solution was centrifuged at 5000rpm.Phenol/chloroform/isoamyl alcohol (25:24:1)was added to the superna-tant,and the mixture was shaken for 20min and then centrifuged at 5000rpm.Cold ethanol was added to the supernatant and the precipitated DNA was washed with 70%ethanol.After drying of the precipitate,the DNA was suspended in TE (10mM Tris –HCl,1mM EDTA [pH 8.0]).The DNA concentration was calculated on the basis of absorption (A 260of 1.0=50mg/L)after measuring the absorbance at wavelengths of 260and 280nm.The purity of the DNA was judged to be acceptable on the basis of the criterion:A 260/A 280N 1.75.RNA contamination was tested by examining an aliquot of the extracted DNA by agarose gel electrophoresis.
Preparation of specific probes used for Southern blot and dot blot analyses A probe specific for the heavy-chain coding region was used for Southern blot analysis,whereas a probe specific for the light-chain coding region was used for dot blot analysis.
Fluorescence labeling of the DNA probe DNA was labeled with the ECF random primer labeling module (GE Healthcare,Princeton,NJ,USA)in accordance with the standard procedure described below.DNA (50ng)in 34μl of distilled water was heated to 95°C for 5min and chilled on ice for 2min.Nucleotide mix (10μl),the solution of primers (5μl),and enzyme solution (Klenow,1μl)were added to the DNA solution and the mixture was allowed to react overnight at 37°C.The reaction was stopped by adding 2μl of 0.5mol/L EDTA (pH 8.0).After the reaction,the DNA was denatured by incubation at 95°C for 5min,then chilled quickly on ice and used as the specific probe for hybridization.
Southern blotting Chromosomal DNA extracted from the cells was digested with the restriction enzymes Pst I,Nco I,and Hinc II.Ten units of restriction enzyme was added per 1μg of DNA,and the DNA was digested at 37°C for 2to 4h.Digested chromosomal DNA was subjected to electrophoresis in an agarose (1%)gel for 3h at 80V in electrode buffer (40mM Tris-acetate,1mM EDTA).After electrophoresis,the DNA was denatured by immersing the gel in denaturing solution (1.5mol/L sodium chloride,0.5mol/L sodium hydroxide)with gentle shaking for 30min at room temperature.The gel was next mounted on filter paper (Whatman 3MM,Whatman Inc.,Clifton,NJ,USA)wetted with transfer solution (1.5mol/L sodium chloride,0.25mol/L sodium hydroxide),and a nylon membrane (Hybond N+)was placed on the gel.The DNA was transferred to the membrane by capillary blotting overnight.The membrane with the DNA was washed with 2×SSC (0.3mol/L sodium chloride,0.03M sodium citrate).The membrane was air-dried and the DNA was cross-linked by UV illumination for 5
min.
FIG.2.Time course of cell proliferation and cell viability (A),and mAb concentration in broth (B)during cell culture.Solid symbols represent Lot 1data and clear symbols represent Lot 2data.Squares represent viable cell density and circles represent cell viability.Triangles represent mAb concentration in the broth.
TABLE 2.Doubling time,cell specific productivity,glucose consumption and lactate
production at Day 3in production.Lot no.Days Scale (L) D.T.a (h) C.S.P.b (pg cell ?1day ?1)Glc c (g/L)Lac d (g/L)13160022.830.4 1.380.622
3160025.434.90.920.59
a Doubling time.
b Cell-specifi
c productivity.c Glucose consumption.d
Lactate production.TABLE 3.The effects of re-activation of cells harvested from the second production
batch.Culture date Days Scale (L) D.T.a (h)Viability (%)
Day 53
00.1–83.5Days 53–5410.1ND b 65.9Days 54–5730.125.395.9Days 57–6030.127.096.3Days 60–6440.132.490.7Days 64–67
3
0.1
25.6
95.6
a Doubling time.b
Not determined.
TABLE 4.MTX tolerance tests and L-Proline auxotrophic tests for EPC2.
Tests Days Scale (L) D.T.a (h)Viability (%)
C.S.P.b (pg cell ?1day ?1)
MTX +30.134.897.732.4?30.130.297.733.0Proline
+30.124.798.4ND d ?
3
0.1
NG c
73.6
ND d
a Doubling time.
b Cell-specifi
c productivity.c No growth.
d
Not determined.
276KANEKO ET AL.J.B IOSCI .B IOENG .,
Dot blotting The chromosomal DNA was digested with 10units of Bgl II per 1μg DNA by incubating at 37°C for 2to 4h in Bgl II reaction buffer.Standard DNA solutions at concentrations of 1000,500,250,and 125ng/100μl were prepared by diluting the restriction enzyme-treated chromosomal DNA of the pre-production cells with restriction enzyme reaction buffer.The solution of restriction enzyme-treated chromosomal DNA from end-of-production cells or late-expanded cells was diluted to prepare sample DNA solutions with a DNA concentration of 500ng/100μl.The standard DNA and the sample DNA were heat-denatured at 95°C for 5min and then chilled on ice for 2min.The denatured DNA was then transferred onto a nylon membrane by using the dot blotting apparatus,and each well was washed with 100μl of 2×SSC.The membrane was placed for 1min on filter paper wetted with denaturing solution and then immersed in neutralizing solution (1.5mol/L sodium chloride,1mmol/L EDTA,0.5mol/L Tris –HCl [pH 7.4])for 1min.The membrane was air-dried and the DNA was fixed by UV illumination for 5min.
Hybridization and detection of signals Hybridization buffer (5×SSC,0.1%w/v SDS,20-fold diluted liquid block [GE Healthcare],5%w/v dextran sulfate)and the membrane with the fixed DNA were placed in a hybridization bottle and pre-hybridization was performed for approximately 1h at 60°C by rotating the bottle.Hybridization was performed at 60°C overnight after addition of the fluorescently labeled DNA.At the end of hybridization,the membrane was washed to remove any non-specifically adsorbed DNA probe.For Southern blot analysis,the membrane was washed once with 1×SSC,0.1%SDS and then with 0.5×SSC,0.1%SDS,at 60°C for 15min in each instance.For dot blot analysis,the membrane was washed once with 1×SSC,0.1%SDS and then with 0.5×SSC,0.1%SDS,at 60°C for 15min in each instance,and then further washed twice with 0.1×SSC,0.1%SDS at 60°C for 15min.The washed membrane was immersed in buffer A (100mmol/L Tris buffer,300mmol/L sodium chloride,pH 7.5)for 1min and then blocked with 10%liquid block diluted with buffer A for 1h at room temperature.The membrane was reacted for 1h at room temperature with 1/10,000th of anti-fluorescein alkaline phosphatase (AP)-conjugated Fab fragments (Roche Diagnostics,Basel,Switzerland)diluted with buffer A containing 0.5%w/v bovine serum albumin.The membrane was washed for 15min at room temperature four times with buffer A containing 0.3%v/v Tween-20to remove un-reacted antibody.An appropriate amount of Attophos (Roche Diagnostics),a substrate of AP,was applied to the membrane,which was then incubated for 1h.The signal was detected using a FluorImager 595(GE Healthcare).Detection conditions were:laser wavelength:488nm;filter:570DF30;photomultiplier voltage:550V).
Analysis of intensity of dot signals The dot signals detected were analyzed with the image analysis software ImageQuant (GE Healthcare).The dot signals were circled on the screen.The 3D-accumulated values were calculated from the pixel
intensities of the circled spots.The background value was obtained by calculating the 3D-accumulated value of the pixel intensity for a uniform area of the membrane covered with circle the same size as the dot circles.The signal intensities used for the analysis were calculated by subtracting the background value from the dot signal values.
Quantitative PCR The copy number of pre-production cells was determined by quantitative PCR (qPCR)with a 7900HT PCR system (Applied Biosystems,Foster City,CA,USA).The target gene analyzed was IgG1.The standard curve was analyzed in triplicate samples.
RESULTS AND DISCUSSION
The preparation histories of the two batches of end-of-production cells (EPC1,EPC2)and one batch of late-expansion cells (LEC)that we prepared are shown in Fig.1.After thawing frozen stock cells at 37°C,cultures were cultivated in a 100ml spinner flask.Cultivation was started with an initial cell density of 2.80×105cells/ml and a cell viability of 95.1%.The culture volume was gradually increased from a 100ml spinner flask through a 500ml spinner flask to a 160-L bioreactor by dilution and transfer to the next volume every 3to 4days.The target initial cell density was set at approximately 1.0to 2.0×105cells/ml.By this method,a total of 10.8-L of culture broth (a total of approximately 1.32×1010cells)was obtained in the 15-L bioreactor after 36days of subculture and transfer.This culture broth was used to inoculate the 160-L bioreactor by aseptic transfer through a pipeline.The cells were maintained by repeated batch culture in the 160-L bioreactor.After 3days of subculture in the 160-L bioreactor,production culture was started by inoculating the cells into the 1600-L bioreactor.After 7days of Lot 1production culture,the cells were subcultured for 11days in a 100ml spinner flask in order to reactivate the cells and prepare EPC1.After 7days of Lot 2production culture,the cells were subcultured for 14days in a 100ml spinner flask in order to reactivate the cells and prepare EPC2.In total,EPC1cells were cultivated for 18days whereas EPC2cells were cultivated for 21days without MTX selection.
During the seed cell culture period (from medium exchange after cell thawing to cultivation in the 160-L bioreactor through expansion culture),the cells were stable as demonstrated by the average doubling time of 27h,average cell viability of 96%,and average cell-specific productivity of 38pg cell ?1day ?1(Table 1).
Cell growth,cell viability,and mAb concentration in the culture broth during the two lots of cell culture in the 1600-L bioreactor are shown in Fig.2.There were no substantial differences between the Lot 1and Lot 2cell cultures.In other words,age had no significant effect on final product yield.In addition,there were no substantial differences in other parameters investigated (not only doubling time and cell-specific productivity,shown in Table 2,but also glucose consumption and lactate production,shown in Table 2).These results showed that the cells were stable during cultivation in the 1600-L bioreactor.
At the end of cell culture in the 1600-L bioreactor,cells were harvested aseptically and reactivated in a spinner flask.The results of the reactivation of cells harvested from the second lot are shown in Table 3.Cell viability after reactivation in the 100ml spinner flask was 83.5%at Day 53.However,upon replacement of the old medium with fresh medium after 1day,the cell viability increased to 95.9%(Days
TABLE 5.Scale-down cell culture.
Culture date Days Scale (L) D.T.a
(h)Viability (%)
C.S.P.b (pg cell
?1
day
?1
)
Days 53–5740.134.792.647.8Days 57–6040.131.796.733.6Days 60–6440.124.797.628.7Days 64–6730.127.797.326.5Days 67–6810.1ND c 97.4ND c Days 68–7240.129.493.325.8Days 72–7530.124.896.530.0Days 75–7830.122.697.726.0Days 78–8240.122.498.726.6Days 82–8530.136.097.430.1Days 85–8940.124.698.225.4Days 89–9230.137.798.230.7Days 92–9640.127.296.628.4Days 96–9930.126.796.130.1Days 99–10230.121.798.125.3Days 102–10640.120.399.425.3Days 106–10930.122.399.225.0Total average ––27.297.129.1SD d
–
–
5.4
1.8
5.6
a Doubling time.
b Cell-specifi
c productivity.c Not determined.d
Standard
deviation.
FIG.3.Probe and restriction enzyme sites in the transgene.P:Promoter;pA:poly A.
LONG-TERM CHO CELL STABLITY DURING LARGE-SCALE CULTIVATION 277
V OL .109,2010
54–57).We confirmed that cell viability recovered with several subcultures.EPC2were examined for MTX tolerance and L-proline auxotrophy.MTX tolerance test results (cell culture period 3days)are shown in Table 4.In the medium with MTX [MTX(+)]the average doubling time was 34.8h,but in the medium without MTX [MTX(?)]the doubling time was 30.2h (n =3average).The average cell-specific productivity in the medium with MTX was 32.4pg cell ?1day ?1,but in the MTX(?)medium it was 33.0pg cell ?1day ?1.These results show that addition of MTX to the medium had no substantial effects on the doubling time or cell-specific productivity of EPC2.In other words,the EPC2retained their MTX tolerance from the cell-thawing stage to the end of production.The results of L-proline auxotrophy tests of EPC2are shown in Table 4.In the medium with proline [proline(+)],the cell doubling time was 24.7h (n =3average).However,in the proline (?)medium,the concentration of the cells did not double throughout the cultivation period.The cell viability in the proline(+)medium was 98.4%,but that in the proline(?)medium decreased to 73.6%.These results confirm that EPC2was auxotrophic for proline.
The results of the scaling-down of cell culture for preparation of LEC are shown in Table 5.Cells harvested from the 160-L bioreactor were subcultured for 17passages in a 100ml spinner flask.During the scale-down cell culture from the 160-L bioreactor,over the 17
passages in the 100ml spinner flask there was stable cell growth with an average doubling time of 27h,average cell viability of 97%,and average cell-specific productivity of 29pg cell ?1day ?1(an appro-ximately 20%decrease in cell-specific productivity compared with that in Table 1)(Table 5).Furthermore,during the last three subcultures (Days 99to 109)there were decreases in the cell doubling time (20to 22h)and cell-specific productivity (25pg cell ?1day ?1).Genetic stability tests on LEC were performed with EPC1and EPC2.Genomic DNA of LEC was prepared from cells harvested on Day 109.The positions of the probes and restriction enzyme sites used in the Southern and dot blot analyses are shown in Fig.3.In the Southern blot analysis with the heavy chain-specific probe,digests prepared using three restriction enzymes,Pst I,Nco I,and Hinc II,were predicted to give bands of ca.5.6kbp,ca.2.2kbp,and ca.1.4kbp,respectively.The results of Southern blot analysis using the heavy chain-specific probe and three restriction enzymes are shown in Fig.4.There was no substantial difference in the DNA restriction fragment pattern between the long-term cultured cells (EPC1,EPC2,and LEC)and the pre-production cells (control).The expected bands were detected in the control genomic DNA and long-term cultured genomic DNA,indicating that there were no deletions or insertions within the ca.5.6-kbp region containing the heavy-chain,DHFR,and light-chain genes (Fig.3).This confirms that the DNA of long-term cultured cells had the same restriction enzyme cleavage pattern as the control genomic DNA.The above results confirmed that mAb-producing r-CHO cells were genetically stable during long-term culture.To assess the stability of the copy number during long-term culture of mAb-producing r-CHO cells,dot blot analysis was performed using genomic DNA from EPC2,LEC,and pre-production cells (controls).The results of the dot blot analysis are shown in Fig.5.The ratios of the signal strength of 500ng EPC2and LEC DNA to those calculated from the calibration curve for 500ng pre-production-cell DNA were 0.87and 0.97—nearly 1.0(Table 6and Fig.5).This result suggests
that
FIG. 4.Southern hybridization using the heavy chain-specific probe and three restriction enzymes.1,5,9:pre-production cells;2,6,10:EPC1;3,7,11:EPC2;4,8,12:
LEC.
FIG.5.Dot blot hybridization.1,3:pre-production cells;2:EPC2;4:
LEC.
FIG.6.Relationship between doubling time and cell-specific productivity.The solid dot represents the 0.1-L-scale data scaled-down directly from the 160-L vessel (Days 53to 57).Clear circles represent 0.1-L-scale data from the subculture from Days 57to 109.
278KANEKO ET AL.J.B IOSCI .B IOENG .,
there was no difference in gene copy numbers between the pre-production cells and the long-term cultured cells.Therefore,the mAb gene in r-CHO cells was stable during long-term culture.These results confirmed that the mAb-producing r-CHO cells were genetically stable during long-term cell culture.Instability of protein production is a common problem in CHO expression systems,and is often attributed to loss of genes encoding the targeted protein (14–17).In addition to the loss of genes encoding the targeted protein,the appearance of non-producing cell populations has been reported (18–21).This event was not observed during our study.The observed decrease in cell-specific productivity with increasing cell growth rate (observed in long-term cultivation in the small-scale vessels,i.e.spinner flasks;see Table 5)was further investigated (Fig.6).The relationship between doubling time and cell-specific productivity in the spinner flasks is shown in the figure (correlation coeffi-cient=0.681),and the closed symbol represents the scaled-down point directly from the different-sized culture vessel.These para-meters were influenced by environmental conditions such as scale-up stress and scale-down stress.Cells in spinner flasks undergo less stress than cells in large-scale bioreactors because,for example,there is less shear stress in the small flasks (22).Under the low-stress conditions of the spinner flask,it is possible that essential endogenous genes such as housekeeping genes remain active,but exogenous genes such as insert genes are silenced.The silencing of exogenous genes is not immediately apparent in the spinner flask because the high stress during cell thawing persists for some time during the spinner flask culture.The gene expression level decreases gradually as the cells adapt to this environment.
We assessed long-term cell stability,especially genetic stability,on a small scale.However,we had not predicted that the transcription level of the exogenous gene would be controlled by the sensitivity of the cells to stress in the culture environment.This suggests that a moderate stress maintains the balance between cell growth and metabolism for production,but that unbalance is induced under low-stress culture environment such as spinner flasks.This leads to a trade-off between cell proliferation and cell-specific productivity (see Fig.6).
In conclusion,we confirmed that CHO cells are stable for long-term and large-scale antibody production.This is the first report on the stability of CHO cells in large-scale bioreactors.CHO cells are now the most commonly used cell line,in combination with DHFR-selectable and amplifiable markers.A number of reports of MTX-amplified CHO cells producing recombinant proteins have shown that such cells are unstable for long-term cultivation with or without MTX,but not to the same extent as CHO cells in the absence of the selective pressure of MTX (23,24).The r-CHO cell line used in this study has low copy numbers of recombinant genes under low selection pressure and single cell cloning.Therefore,loss of recombinant gene copy number caused by genetic rearrangement and the appearance of non-producing cells as a result of diversification of the cell population were not observed.It is important to note that long-term cell culture in the same culture vessel had a substantial effect on cell proliferation.The doubling time was short when the maximum viable cell density was high in the subculture.A decrease in cell-specific productivity was associated with an increase in the rate of cell proliferation.This means that there was a trade-off between cell proliferation and cell-specific productivity.There were no substantial differences in the
genetic characteristics of the cells or any other parameters studied.These results indicate that the cells were stable during long-term production of recombinant mAb.This report indicates the possibility of robust monoclonal antibody production using repeated-batch-culture systems;this has already been successfully applied at a practical scale.In general,as part of the production of therapeutic proteins,cell lines must be frozen at various stages to create cell banks.According to Barnes et al.(25),cryopreservation and revival procedures do not alter the stability of NS0cell lines.We also have data that show that cryopreservation and revival procedures do not alter the stability of CHO cell lines (data not shown).Therefore,vials of r-CHO cell banks can be used to produce mAbs with reproducible results.Furthermore,based on the results of the present study,one vial can be used to produce many batches (lots)of recombinant proteins.
ACKNOWLEDGMENTS
The authors wish to thank the entire team at Chugai for their support during this work.References
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TABLE 6.Copy number ratio of long-term cultured cells to pre-production cell.Sample
Copy number ratio
Copy number
Pre-production cells 123.6a EPC20.8720.6b LEC
0.97
22.9b
a Determined by quantitative PCR.
b
Calculated by copy number ratio to pre-production cells.
LONG-TERM CHO CELL STABLITY DURING LARGE-SCALE CULTIVATION 279
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280KANEKO ET AL.J.B IOSCI.B IOENG.,
中国梦我的梦演讲稿范文5篇
中国梦我的梦演讲稿范文5篇 篇一 亲爱的老师、同学们：一个人梦想的追寻和实现，成就了中国的腾飞和光荣，这就是“中国梦”。 比如：神州五号载人飞船的发射成功，嫦娥三号圆满发射，都是中国富强的表现。曾经，邓小平说过：”我要让中国富起来!“还有周恩来总理：”我要让中国强大起来!“后来他们都实现了自己的中国梦。然而，我的中国梦就是——让中国变成绿色。 现在，因为伐木工过度砍伐树木，水泥厂越来越多，沙漠化越来越厉害，还有农业污水越来越多，把蓝色的小溪都变成了灰色和黑色。中国的环境越来越差，全是灰色和黄色。我实在是太心疼了，不忍心看下去，所以我逐渐的开始保护环境。在过年的时候，我会对爸爸妈妈亲戚朋友分别说：”今年不要放烟花爆竹了，您看那灰色的天空，还有电视上说的雾霾天气，环境多差呀!“有空的时候，我还会出去打扫卫生，甚至植几株草和花。植树节，我还会去植树。我还会有空的时候去贴几张”告示“，让大家不要破坏花草树木等。每个普通中国人的普通中国梦，都可以汇聚成一个伟大的中国梦!如果每个人都实现了自己的中国梦，那么我们的中国会越来越强大。如果每个人都嘴上说说，却不去实现，口是心非，那又有什么用呢? 篇二 亲爱的老师、同学们：每个人都有梦想，老师有希望学生成绩出色的梦，军人有希望国家繁荣富强的梦，医生有希望病人早日康复的梦，宇航员有希望发星球更
多秘密的梦…… 同样，我也有一个美好的梦。我梦想长大以后当一位能歌善舞的舞蹈家，代表国家四处比赛，为祖国争光。为了实现这个梦想，每周，我都要去文化馆上舞蹈课，上课前，我们都会自己拉拉腰、压压腿，做好课前准备，以免上课时把身体拉伤。虽然做了课前准备，可在练习时，总会清晰地听到自己骨头咔咔响的音。有时把一只腿放在厚厚的毯子上，另一只腿放在地上，摆成竖叉，大腿贴地，然后用手拉住后面那只脚，倒数二十秒，就可以了。可是，在这过程中，总会感到疼痛难忍，有时，我会疼得哭起来，有时，会疼得坐不稳，每当这时候，只要心里一想到那个美丽又美的梦，我就不觉得那么痛了，也轻松多了。有时在排练舞时，有的动作很难，练了很多次，可还是没做好。每当我相放弃不练了的时侯，只要一闭上眼睛，脑海里就会浮现出我在台上表演，台下的观众个个鼓掌叫好的场景。这些景象让我一次又一次地从摔倒的地方重新站了起来，完成了一个又一个困难却好看的舞姿。每当我们表演完节目，崔老师总不忘鼓励我们一句‘你们今天表现得真棒!’虽然这话在别人听起来十分平淡，可我听起这话总能让我紧张不以的心平静下来，感受到温暖。通过自己的努力，我参加了许多比赛，每次拿到奖时，我都十分激动，心里像吃了蜜一样甜甜的，希望自己更上一层楼。 也许，在不久的将来，我的梦想会成为现实，我一定要好好学习，天天向上，努力实现我美好的理想。 篇三 亲爱的老师、同学们：梦想是什么?相信每个人都有自己的理解，是卖火柴的小女孩渴望点燃一根火柴来取得温暖，还是丑小鸭幻想自己有一天可以脱去丑陋的
最新版中国梦我的梦演讲稿10篇
最新版中国梦我的梦演讲稿10篇 忆往昔峥嵘岁月，看今朝锦绣河山。回味改革开放的三十年，华夏神洲留下了太多的梦想奇迹。1997年7月1日，香港正式回归祖国，举国欢庆，“统一梦”迈出了坚定的第一步;2001年12月11日，中国正式成为世界贸易组织成员，从此，国家经济快速、平稳、健康的发展，“小康梦”已不再是一个遥不可及的目标;2013年6月12日，神十问天，璀璨的星河中留下了炎黄子孙的身影，编织了千年的“飞天梦”也终于迎风远航……一个个梦想的实现，一份份成就的辉煌无不昭示着“中国梦”——“复兴之梦”必将在党的领航下乘风破浪、直挂风帆。 走过历史的长廊，回顾百年的沧桑，“中国梦”一词在岁月的书卷中熠熠生辉。作为一种最为朴素、浓烈的民族情感，它的萌发、形成之于耻深痛极的近代百年，有着深刻的时代烙印。无论是谭嗣同“四万万人齐天下泪，天涯何处是神洲”的哀恸，还是秋瑾“拼将十万头颅血，誓把乾坤力挽回”的决绝，所体现的都是对国家前途命运的关切与焦虑。而今，随着时代的变迁，“中国梦”也从“救亡图存”变为了“强国富民”。然而我坚信有一点却始终不会改变，那就是坚定的民族自信，国人始终坚信有着五千年优秀历史文化的中华民族，无论面临怎样严峻的考验，终将转危为安、再
造辉煌，实现百年的“复兴之梦”。 俗话说：“聚沙成塔，集腋成裘”。“中国梦”既承载着国家的强国之梦、民族的复兴之梦，也包涵着亿万国人同胞千千万万个小小的梦想。有“富裕梦”，有“健康梦”、有“求学梦”……正是由于这些如涓涓细流般的梦想汇聚成河，才会让“中国梦”愈发的凝实。大学毕业之初，怀揣着对未来的憧憬，我选择了公务员考试。最终，经过重重选拔、层层考验我来到了隆化这个美丽的小城。抛弃了国企的高薪，远离了父母的怀抱，背离了自己的故乡，踏上一个陌生城市的土地，在这里去拼搏、去奋斗，这样的生活真的是我想要的吗我也曾无数次的扪心自问。时至今日，月余的法庭生活给了我一个满意的答复。这里也许没有车水马龙的的喧闹，却有着一份乡村独有的宁静;这里也许没有灯火通明的高楼，却能映照出一张张质朴的面容;这里也许没有频繁的人际交往，却有着一句句真诚的关怀。在这片广袤无垠的大地上，我找到了追寻梦想的原动力。 选择了到基层工作就意味着选择着艰苦;选择了公务员这个职业就意味着肩负着责任;选择了到法院任职就意味着守护着公正。在过去的一个月里，我体验到了从一个懵懂的书生向一名法院工作者转变的酸甜苦辣。现实和理想尽管错位不断，但热情和动力却与日俱增。基层法庭是法院工作的前沿阵地，直接面向农村，密切联系群众，受理的案件虽不
为实现中国梦做贡献 小学生征文
向着中国梦，前进！ 绥芬河市北寒小学四年级李鸣 梦，通常指人们的理想。每个人都会做梦，每个人都会有自己的梦想。美羊羊的梦想是能够在青青草原上尽情地玩耍，光头强的梦想是把大森林里的树木都砍光。我的梦想，可以是一个漂亮的发卡，一盒可口的点心，是考试后的100分，妈妈对我夸奖的笑容……可今天，老师说，我们每个人不但要有自己的梦，还要有中国梦。 那么，什么是中国梦呢？中国梦是建立在祖国发展基础上的，是对祖国有利的梦，每个人的中国梦都不相同，有大有小，但它们又都有相同之处，那就是都怀着一颗为祖国作出贡献的心，对人民有功利的心。我想中国梦还应该是国家富强，小朋友们都有新衣服穿，有变形金刚、芭比娃娃；民族团结，不会有战争与饥饿；大人们高高兴兴地上班，孩子们能够一块儿在草地上做游戏。但我觉得，最重要的是要建设一个美丽、洁净的家园，那就是天蓝、地绿、水清、气爽的美丽中国。没有地沟油，没有沙尘暴，没有禽流感…… 建设美丽中国，就要从小事做起。在学校，看到地上有纸屑，我会主动弯腰捡起；看到水龙头滴水，我要主动上前把它关紧；看到有人说脏话，我会及时上前制止。春天来了，我拉着爸爸妈妈买树苗，到附近山坡上植树，因为我知道，绿化造林是防治大气污染的一种有效方法。我还要宣传环保知识，动员更多的人加入环保队伍，让天变得更蓝，水变得更绿。我还会在村里钉上一块“爱护环境、人人有责”的警示牌，让全村的人跟我一起行动起来，保护我们的环境、净化我们的环境。我知道我一个人，一家人的力量很微薄，但是如果全中国的孩子都能行动起来，每个中国家庭都行动起来，那就会汇成强大的环保能量，才能建设好美丽中国。 每个中国人都有着自己的中国梦，只要每个人能为这伟大的梦想贡献出自己的力量，哪怕是微不足道，但当所有的贡献汇集到一起，将会是一股多么强大的力量，这股力量会把中国推向更美好的明天，只要人人行动起来，我相信建设美丽祖国这个中国梦将会很快实现。
中国梦-我的梦演讲稿
中国梦我的梦 有一种东西，它承载着人们的希望，虽然他看不见，摸不着，却能在人们心中产生巨大的力量。它，就叫梦想。上帝没有赐予我们翅膀，却赐予了我们会飞的心灵和能够梦想的大脑，使我们有了一双“隐形的翅膀”，带着我们在人生辽阔的天空里自由地翱翔。 今年三月，全国人大和全国政协会议在北京召开，关于“中国梦”的话题也进行过热烈的讨论。每个人或多或少也在思考“中国梦”的问题。 那么，中国梦是什么呢？习近平总书记说“每个人都有理想和追求，都有自己的梦想。实现中华民族的伟大复兴，就是中华民族近代以来最伟大的梦想。这个梦想，凝聚了几代中国人的夙愿，体现了中华民族和中国人民的整体利益，是每一个中华儿女的共同期盼。”是的，每个人都有自己的梦，梦想能够照亮生活，亦能成就未来。而有一个梦，它既是你的梦，也是我的梦，它是万千中华儿女共同的心愿，那就是实现中华民族伟大复兴的中国梦。 每个人的梦都是中国梦的组成部分，我的梦亦如此。都说学生的主业是学习，的确如此，我的梦来自学习。我喜欢在午休时一个人独自读书的静谧；喜欢在难题前不停运用各种公式演算的执着；也喜欢与同学一起为一道题目争执不休的吵吵闹闹。这样所诠释的梦是理想之梦。 梦也来自家庭，对于我的成长，它最有发言权。平日里与父母相处的时间虽然不多，但是一起吃饭时，父亲在饭桌上的高谈阔论；
坐在同一书桌前各自忙碌时，偶尔抬头与母亲随意交谈的闲话，都在影响着我对生活的态度。 有了梦，便要去追寻。三年前，在懵懂的状态下，在家长的引导下选好了脚下的路—上高中、考大学。渐渐地，考大学也真正成为了我的梦。清晰地记得，老师在中考前说“我们将做出人生第一次重大选择”，面对众多的学校，我固执地选择了朝中，心中似乎在坚守着什么。后来，随着一个个目标的制定，无数次为“自己的事”的奋斗，我逐渐明白，我是在坚守着自己的梦，并且开始懂得为了理想和追求而努力拼搏。其实走到现在，蓦然回首，才发现人生是一个条件从句，梦的方向并无过多区别，而我们最终选择的那个梦，正是因为被自己选择，被自己填充，才显得与众不同，才显得适合自己。 我相信，只要坚持不懈，努力拼搏，梦想就一定能够成真。我的梦，我们的梦，铸就了实现国富民强的中国梦。可以想想，如果中国民族实现了伟大复兴，那么世界梦一定会更加流光溢彩。
中国梦我的梦演讲稿
演讲稿一 尊敬的各位领导、敬爱的老师、亲爱的同学们： 大家好!今天我演讲的题目是《中国梦，我的梦》。 在发言前我想问同学们一个问题就是：你们有梦吗?假如有梦的同学请大声的回答我。很好!我想你们的梦一定是绚烂的、美丽的、丰富的、美好的。生命因责任而美丽，人生因梦想而精彩。假如还在犹豫自己真正的的梦是什么?请认真听我们中国所共同的梦——中国梦 人生如梦，梦想是帆，每个人都有一个只属于自己的梦，但我们同属一个国家，所以每个人的梦又与国家的兴衰荣辱紧密相连。先哲顾炎武曾说：“天下兴亡，匹夫有责。”只有国家好，大家才能好。 有梦才能使中国富强! 我依然清楚的记得： 当甲午战争战败，日寇无礼踏破中国的门户;当八国联军侵入北京，无情掠夺中国的财产;当七七事变发生，中国的老人、妇孺被残忍杀害的时候，我在想那时中国的梦是怎样的! 我虽不曾亲眼看到，但那却是铁一般的事实。因为从老人们那深邃的眼神中可以感到无尽的愤懑;从他们干瘪的脸颊可以看到深情的泪水，从他们嘹亮的军歌中可以想到那奋勇杀敌时的豪迈;从他们激昂话语中听到那誓要捍卫家园振兴中华的誓言。作为新一代青年的我们难道不应该树立远大的理想，付之以踏实的行动，去继承先辈们的使命。去实现中华民族的伟大崛起和复兴吗? 有梦才能使中国繁荣! 在改革开放以来中国取得了一系列的可以载入中国史册的成就。香港、澳门的回归，经济特区的建立，使中国成为发展国家中的经济大国，科技先进国和军事强国。当中国成功举办奥运的时候，当神九飞天的时候，当蛟龙入海的时候，当航母下水的时候，当莫言荣获诺贝尔文学奖的时候。我相信每个人都感觉到了无比的自豪。但是现在的中国与其他发达国家还有很大差距。作为新一代的我们，难道不应该志存高远吗? 我想有的人会说，我们的力量是有限的。的确个人的力量很渺小，但是中国梦就是因一个个微不足道的个人的梦一直汇集、汇集，然后凝聚成的一个巨大的梦。冯至在《十四行诗》中写道，我们准备着，深深领受，那些意想不到的奇迹，在漫长的岁月里，忽然有彗星的出现，狂风乍起。 梦想是美丽的，它是最美的期望;梦想是阳光的，它使人由浮躁走向踏实;梦想是充满力量的，它可以激发人身体里无限的潜能。我们期盼的是国泰民安、经济发展、政治清明、文化繁荣、社会和谐、生态良好、公平正义。这才是中国人最伟大的梦。
2018年工信财务处我为实现中国梦做贡献演讲稿
2018年工信财务处我为实现中国梦做贡献演讲稿 梦想在前路在脚下 梦想，是每个人希望的开始。中华民族五千年历史传承着一个长长的梦，几经辗转，几经沉浮。时至今日，汇聚成了一个梦，中国梦。 一个人不能没有梦想。因为有梦想，我们才经历坎坷依然前行，因为有梦想，我们才经历沧桑信心不改。有梦想才会有希望；有希望才会有激情；有激情才会有事业；有事业才会有未来。中华民族是一个命运共同体，只有民族、国家全面科学发展，个人才能实现梦想。同样，只有每个人都充满激情和梦想，“中国梦”才够美丽，才够坚实。 工信人同样承担着实现中国梦的使命，一代一代在工业和电子、信息产业的人们前仆后继，奋勇拼搏，为了这个梦想奉献着自己的青春。 “疾风知劲草，岁寒见后凋”在财务处工作这段时间以来，我有过艰辛的汗水、也有过收获时的喜悦；有过扬帆起航时的热忱与豪情、也有过风雨骤来时的动摇与退却；有过风平浪静时的欢喜与惬意、也有过漫漫征途的寂寞与煎熬！财务工作赋予我人生新的意义和追求，它为我带来了淳朴的友谊，让我品尝到工作的无限快乐。在梦开始的这片热土上，我们平凡的财务人员在平凡的岗位上，默默无闻，奉献青春；我们用坚定的信念，用点滴的小事，书写着一幕幕爱岗敬业的篇章。 我们财务处就是这样一个群体，整日跟毫无生气的数字凭证、账簿打交道，整理核算杂乱的原始票据和凭证，日复一日，与数字相伴，与票据为友，但这一切都磨灭不了我们对财务工作的饱满热情，我们无怨无悔。既然扎根于工信财务工作，那么确保数字核算的准确无误，各项财务制度执行的缜密就是我们的责任，按时、顺利、高效地完成会计核算工作，为领导及时提供准确数据，为各部门提供最好服务，为厅机关及厅系统各项工作的顺利开展提供有力的保障是我们每个财务人员最大的心愿和使命！ 梦想不是说说而已……我有一个大大的梦想，那么这个大大的梦想要从现在小小
关于我的梦中国梦主题演讲稿800字
关于我的梦中国梦主题演讲稿800字 尊敬的各位老师： 你们好!今天我演讲的题目是《我的梦中国梦》。 我有一个梦想，深深扎根于我的心中。那就是长大后，我要成为一个科学家。 尽管我没有过人的才智，没有严密的思维，也没有特别准确的判断力，但是我仍不会放弃努力。尽管这个梦想距我很遥远，但我仍 不会停止追求。尽管在实现梦想的过程中，会有很多挫折和无数的 磨难，但我仍不会灰心丧气。因为我相信，只有经历地狱般的磨练，才能练出创造天堂的力量;只有流过血的手指，才能弹出世间的绝唱;只有经历困难和挫折，才能实现自己的梦想。 以前，每当我看到科学家们令人瞩目的成就时，总会感到羡慕和敬佩。是他们，推动了社会的发展;是他们，使人民生活水平得到提高;更是他们，为祖国的发展赢来了一个崭新的明天。 因此，我想成为一个科学家，成为一个对国家有贡献的人，成为这个国家的栋梁。每当我看到浪费时间的人时，我会为他们感到惋惜;每当我看到灰心丧气的人时，会为他们感到悲哀;每当我看到不 务正业的人时，我会感到愤恨。因为他们没有看到自己的价值，没 有属于自己的梦想。这样的人生，是没有意义的人生。 而我，至少有一个梦想，一个目标。有了这个梦想，我就会一直努力下去，永不放弃。有了这个梦想，就等于把握了自己的人生航向，不会再迷失方向。有了这个梦想，就好象一盏明灯，照亮了我 前进的道路。一直通往胜利的顶峰。 我有梦，中国也有梦。我的梦想，用自己的智慧站在时代的顶峰，中国的梦，用自己的勤劳，自立于世界之上!为了这个梦想，他发奋，他图强，他忍受无法言语的苦难，只为自己可以挺起胸膛!地震来了，
不怕，他有的是铁一般的脊骨，洪水来了，不怕，他有的是山一般 的胸膛!奥运会来了，不怕，他有的是腾飞的翅膀!有梦的人，才是 真正的人，有梦的国，才是真正的国!我的梦就是国的梦，我的梦， 成为科学家，为国家尽力，国的梦，繁荣富强，让我们幸福! 我的中国梦演讲到此结束，谢谢大家! 中国梦也是我的梦。我生在中国，长在中国，读过中华几千年的历史，曾为之激动，忧伤，悲愤，骄傲，曾为之食不能安，夜不能寐，，中国已与我紧密的联系在了一起，我为中国伤心，为中国欢乐。我还记忆犹新那句话：犯我大汉天威者，虽远必诛。它深刻的 体现出我中华民族，我大汉的骄傲与荣耀，或许很狂妄，或许很嚣张，但它却让我深深为之感动，为了中华的荣耀，我可以吃苦，可 以受累，甚至我可以献上我微不足道的生命，可就是这份愿意，让 我为中国喝彩，让我对中华民族更加钦佩，对中华历史更加热衷。 或许，我说的对;或许，我说的不对，但是我想认同我的人总是有的，我想这么做的人总是有的! 中国梦，到底是什么，我仔细地思考过一番，我想中国梦应该是振兴中华的梦想。虽说我们消沉过几百年，但是我们也荣耀过几千年，所以我们中华民族最不缺少的便是一种资质，一种让我们曾经 荣耀过几千年的资质，所以我不认为我们与其他民族相比有任何的 不如之处，所以我相信认清现实之后的我们必将会取得成功，必将 会实现我们近年来振兴中华的梦想。 霍去病将军匈奴未灭，何以为家。诸葛亮鞠躬尽瘁，死而后已。周恩来为中华崛起而读书...... 这些历史上让人怀念的伟人们，都有一个和中国梦一样的梦。霍去病将军将自己的一切都献给了中华民族的安危与荣耀，虽说他并 不完美，但瑕不掩瑜，他守护大汉，永不后悔，留下了封狼居胥的 佳话。至于诸葛亮很多资料认为罗贯中是亲蜀派将蜀国的人物进行 了美化，比如历史上根本没有他三气周瑜的事情。但是罗贯中为什 么这么写，我个人认为除了刘备得民心之外，还有一点，那就是蜀 汉一称，正因为蜀国在后面加上了一个“汉”，赢得了更多人的偏
为实现中国梦做贡献_小学生征文
向着中国梦，前进！ 学校：南召中心校务滋小学班级：五年级 学生：李斯言 指导教师：屈海川
向着中国梦，前进！ 梦，通常指人们的理想。每个人都会做梦，每个人都会有自己的梦想。美羊羊的梦想是能够在青青草原上尽情地玩耍，光头强的梦想是把大森林里的树木都砍光。我的梦想，可以是一个漂亮的发卡，一盒可口的点心，是考试后的100分，妈妈对我夸奖的笑容……可今天，老师说，我们每个人不但要有自己的梦，还要有中国梦。 那么，什么是中国梦呢？中国梦是建立在祖国发展基础上的，是对祖国有利的梦，每个人的中国梦都不相同，有大有小，但它们又都有相同之处，那就是都怀着一颗为祖国作出贡献的心，对人民有功利的心。我想中国梦还应该是国家富强，小朋友们都有新衣服穿，有变形金刚、芭比娃娃；民族团结，不会有战争与饥饿；大人们高高兴兴地上班，孩子们能够一块儿在草地上做游戏。但我觉得，最重要的是要建设一个美丽、洁净的家园，那就是天蓝、地绿、水清、气爽的美丽中国。没有地沟油，没有沙尘暴，没有禽流感…… 建设美丽中国，就要从小事做起。在学校，看到地上有纸屑，我会主动弯腰捡起；看到水龙头滴水，我要主动上前把它关紧；看到有人说脏话，我会及时上前制止。春天来了，我拉着爸爸妈妈买树苗，到附近山坡上植树，因为我知道，绿化造林是防治大气污染的一种有效方法。我还要宣传环保知识，动员更多的人加入环保队伍，让天变得更蓝，水变得更绿。我还会在村里钉上一块“爱护环境、人人有责”的警示牌，让全村的人跟我一起行动起来，保护我们的环境、净化我们的环境。我知道我一个人，一家人的力量很微薄，但是如果全中国的孩子都能行动起来，每个中国家庭都行动起来，那就会汇成强大的环保能量，才能建设好美丽中国。 每个中国人都有着自己的中国梦，只要每个人能为这伟大的梦想贡献出自己的力量，哪怕是微不足道，但当所有的贡献汇集到一起，将会是一股多么强大的力量，这股力量会把中国推向更美好的明天，只要人人行动起来，我相信建设美丽祖国这个中国梦将会很快实现。 建设美丽祖国——这个伟大而又现实的中国梦，我会行动起来的，爱护环境、节约用水……，我要向着中国梦，前进！
中国梦我的梦主题演讲稿
中国梦我的梦 每当听着国歌，看着鲜艳的五星红旗冉冉升起，我总是热血沸腾，梦想着为国家的富强贡献自己微薄的力量，纵然力量微薄，但只要大家一起为我们美丽的祖国做贡献，就可以使她变得更强更美。梁启超先生说过：“少年富则国富，少年强则国强”，我们年轻人的命运与国家的命运息息相关，同样，强国梦与我们的梦想荣辱与共，实现中国梦，就是我的梦。 实现中国梦就需要我们紧握梦想的缰绳。 实现中国梦需要有正确的方向，找到了方向，勤加努力就会成功；找不到梦想的希望，或者妄自堕落，或者碌碌无为，都会抱憾终生。如果说梦想是马，那么我们就需要抓住梦想的缰绳，找到方向，加速驰骋。爱国和报国的信念就是我们梦想的缰绳，古往今来，多少仁人志士为了爱国报国前仆后继，他们用生命铸成了中华民族的不倒长城，他们用热血染成了中华民族的鲜红旗帜。想起爱国报国，我们仿佛看到岳飞在战场上高举精忠，文天祥在狱中延续丹青，仿佛看到张自忠横刀立马，杨靖宇挥斥峥嵘；先烈们为我们指明了中国梦的方向，我们更应该延续我们的梦想，紧握梦想的缰绳，让爱国之心长存，让报国之心长青，才能让梦想之门长开。 实现中国梦就需要我们握紧梦想的双桨。 实现我们的梦想就需要坚强的意志和毅力，任何道路都会有坎坷和挫折，实现理想的道路更不是一帆风顺，无数的困难和险阻会阻挠
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中国梦，我的梦。"墙角数枝梅，凌寒独自开"，我想做一朵梅花，傲立雪中，不屈不挠，自强不息。曾经为了中考，操场上的挥汗如雨，考场上跃动的笔杆。今日站在这里，成为祁县中学实验班的一员，继续追逐我的大学梦。古人云："行胜于言"。月已高悬，夜已人静，仍有挑灯苦读，思想之花穿行于夜空，几何图案填充星宿，梦的翅膀到我飞行。认真，勤奋，坚持，顽强，把握高中生活，珍惜一点一滴，是我的梦，也是我们的梦。我们用汗水浇灌梦想之花，让它开放，散发芳香。是的，我们正在一步一步的绘着中国梦。试问：少年无梦，国哪有梦？少年无志，国哪有志？少年不强，国哪能强？ 地平线上冉冉升起的太阳，如我们；海上掀起的层层浪花，如我们；那愈燃愈旺的火焰，如我们，我们肩负着中华民族伟大复兴的历史重任。让梦如黑夜中的星星，指引我们前行；让梦如火，照亮前方的路；让梦如光，绽放无限魅力。 当今世界，经济全球化，科技高速发展，竞争力不断加强。中国梦，让中国强大，让中国发展，让中国雄鸡挺起胸膛，让亚洲雄风吹遍世界。为此，让我的梦，让我们的梦，共同点燃中国梦，让我们用知识开启圆梦引擎，用品格筑牢圆梦基石。
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小学生我的梦中国梦演讲稿10篇
小学生我的梦中国梦演讲稿10篇 亲爱的老师、同学们： 大家好! “林花谢了春红。太匆匆，无奈朝来寒雨晚来风。胭脂泪，留人醉，几时重，自是人生长恨水长东。”感叹的是时光的流逝。岁月 的翩然轻檫，无可奈何。“人生若只如初见，何事秋风悲画扇。等 闲变却故人心，却道故人心易变。”是对人事沧桑的感概：“苟利 国家生死以，岂因祸福避趋之。”驾长车踏破。贺兰山缺。壮志饥 餐胡虏肉，笑谈渴饮奴血。这是林则徐和岳飞的爱国宣言，不算激 昂也不算壮阔，却表达了他们深深的爱国之情，在我心里。这些文 化的结晶代表了中国一代人的思想，有的是相思情，有的是人生叹。而有时。却是中国梦。 90后的我们奋发图强，乐以忘忧，青灯黄卷，宵衣肝食，是的，生应无所息。王维曾经在人间词话中说过三重境界;一是“衣带渐宽 终不悔，为伊消得人憔悴”;二是“昨夜西风凋碧树，独上高楼，望 尽天涯路;三是”众里寻他千百度，蓦然回首，那人却在，灯火阑珊处。’在我看来，我们的求学道路，我们的爱国道路，都需要经过 这三重境界，方能上善若水，泽万物而无言。一叶绽放一追寻，一 花盛开一世界。我们的中国梦，是和平，是暖，是希望。是燕在梁 间的呢喃，是人间的四月天。 亲爱的老师、同学们： 大家好! 很高兴能站在这里演讲，今天我演讲的题目是《我的中国梦》。 当浦江的浪花摇落满天繁星，点亮我们身后中国馆敞开的门窗，我们在流光异彩的世博园里，我的梦在长城上生长，这梦想的长城，正在延伸祖先的荣光，长城的脚下，是五千年文明的土壤，长城的
天界，是一个名叫未来的地方，下一个百年，我的梦，中国梦，花开何方，来吧!同学们!请打开心中最美丽的翅膀，这一刻让我们一起飞向北京，在那万里长城之上对话星空，和世界一起分享， 今夜当世博园的灯光相逢长城的目光，我们要在这里集合起所有属于未来的梦想，哪怕只是一道稍纵即逝的流星，也请关注它，也许哪一天就能触发出新世界的曙光，请未来登上长城吧!一起收获中国少年永无止尽的梦想，少年智则中国智，少年强则中国强，我的梦是中国梦，中国的梦是我们的梦，要实现梦想，不单单要靠自己的努力，更重要的需要同伴多给我鼓励，给我帮助，理解，支持，也就是这份理解，支持，最终会实现我们的梦想。坚持成就梦想。 人可有很多梦想，但是可能实现一个就足够了，只不过是刚刚才第一步跌到了，为什麽就不愿爬起来?明天总要面对，明天太阳还要升起，我们如果还想继续走下去的话，那只能换一条路。天空不留鸟飞的痕迹，但我已经飞过，如果我们尽力了，却依然抵达不了梦的彼岸，我们无悔，因为我们至少奋斗过，如果我们付出了，得到的却不成正比的收获，我们无悔，以为至少我们付出过，此刻，唯有向前，唯有向前 小时候，其实变换梦想没有关系，你需要的是不断的去想，不断地去想快乐的事情，其实梦想不必要很大，只需要觉得这很现实，这你能做得到，但第二个是梦想跟眼泪和汗水，是在一起的，假如梦想离开了汗水的眼泪，那就变成乱想，空想。 亲爱的老师、同学们： 大家好! 今天我演讲的题目是《中国梦，我的梦》。 什么是梦?什么是中国梦?历史的点点滴滴如散落在偌大沙滩上的沙石贝壳，我悄悄走过，贪婪地看着这些晶莹宝贵的财富，时而拾起一两颗打动心灵的贝壳，寄出一份梦想，蹲下投放。中国梦，流淌在岁月。
大学生为实现中国梦做出自己的贡献
论大学生为实现中国梦做贡献 随着社会主义经济体制的逐步建立和改革开放的不断深入，我国社会各个领域发生了巨大的变化，对当代大学生的人生价值取向也直接产生了影响。如何进一步加强对当代大学生的思想政治教育，营造积极向上的校园文化，为实现中国梦做出贡献，这成为当前高校思想政治工作的一项重要任务。 中共十七大报告中指出，要建设社会主义核心价值体系，增强社会主义意识形态的吸引力和凝聚力；要切实把社会主义核心价值体系融入国民教育和精神建设全过程，转化为人民的自觉追求。社会主义核心价值体系的提出，为大学生思想政治教育注入了新的血液。大学生作为充满理想、活力和激情的优秀群体，理应成为社会主义核心价值观学习和践行的“先行军”。 一、大学生应培养社会主义核心价值观 核心价值观是一个社会中居统治地位、起支配作用的核心理念，也是一个社会必须长期普遍遵循的基本价值准则，具有相对稳定的特点。社会主义价值观是对社会主义价值的总的看法和最根本观点。在党的十七大召开之前的十六届六中全会上，党第一次提出了社会主义核心价值体系这一科学命题,并对社会主义核心价值体系的科学内涵作了界定。社会主义核心价值体系包括四个方面的基本内容，即马克思主义指导思想、中国特色社会主义共同理想、以爱国主义为核心的民族精神和以改革创新为核心的时代精神、以“八荣八耻”为主要内容的社会主义荣辱观。 青年大学生正处于人生观、价值观形成的关键时期，他们思想观念逐渐趋于成型，但仍具有较大的可塑性；他们接受新鲜事物的能力很强，但鉴别力明显欠缺。赢得青年就赢得未来，我们以社会主义核心价值观加强大学生思想教育，具有鲜明的时代意义和现实意义。应从以下几个方面进行社会主义核心价值观教育： 1.夯实思想政治理论课教学基础。高校思想政治理论课是大学生思想政治教育的主渠道，加强大学生思想政治理论课教育教学，首先科学的课程设置是其基本环节。应充分体现当代中国马克思主义发展的最新成果，全面反映党领导人民建设中国特色社会主义的生动实践和基本经验，全面反映在毛泽东思想、邓小平理论和“三个代表”重要思想、科学发展观指导下哲学社会科学研究的最新进展。 2.提高教师素养，通过教师的言传身教感化学生 教师承担着培育社会主义事业的建设者和接班人的历史重任，对大学生进行社会主义核心价值观教育，高校教师不论是思想政治课教师还是专业课教师要在提高专业知识素质的同时，还要加强政治学习提高政治素质。专业素质是“才”，政治素质是“德”，是教师职业道德中的思想品德。这两个方面是相辅相成，相互促进的。教师加强和提高自身“德”方面的修养，不仅使自己成为一个道德高尚的人和合格的教育者，重要的是通过自己的教育活动和良好的思想、道德、品质、人格把学生培育和感化成德才兼备的人才。 3.树立优秀大学生典型，通过学生感化学生 心理学家认为，人的行为往往被其内在动机和需要所驱动。树立典型，塑造榜样就是为了提供行为参照，确立行为目标。学习者有了目标参照，进而努力使自己的行为与榜样的行为一致。大学生身边的优秀典型，他们的精神比较容易为大学生们所接受。对校园中在思想品德方面涌现的优秀大学生典型要及时大力宣传，使良好的风气在大学校园中弘扬，成为大学校园中主导地位的风尚，使大学生们的行为有可仿效的行为作参照，从而见贤思齐。 二、营造积极向上的校园文化 党的十七大在关于党的建设的部署中，明确提出在党的基层组织和党员中深入开展创先争优活动。探索如何加强校园文化建设以推动创先争优显得尤为重要。校园文化建设是学校稳步发展的保证。校园文化建设的出发点和落脚点是学生。学生是校园文化建设的主体，加强
中国梦我的梦(大学生中国梦演讲稿)
中国梦我的梦（大学生中国梦演讲稿）中国梦我的梦（大学生中国梦演讲稿） 时玉婷 梦是什么？ 从心理学的角度回答，弗洛伊德认为，梦是有意识看无意识的一扇窗子。从医学的角度回答，梦是脑在作资讯处理与巩固长期记忆时所释放出的一些神经脉冲，它是被意识脑解读成光怪陆离的视、听觉所造成的。从梦学的角度回答，梦是人睡眠时的一种心理活动，梦中离奇的梦境是因人睡眠大脑意识不清时对客观事物的刺激产生的错觉引起的。总之，梦是一种主体经验，是人在睡眠时产生的影像、声音、思考或感觉，通常是非自愿的。 那么梦想是什么？ 有人说，梦想等于理想；有人说，梦想就是空想、幻想；还有人说，梦想是期望达到的一种高度。而我说，梦想是心灵的思想，是对美好事物的憧憬和渴望。因此，梦想不是梦。 陈信宏曾说：“如果我不在梦想里，就是在通往梦想的路上。”是啊，谁不是这样呢？每个人都有理想和追求，有人向往自由自在的日子，也有人喜欢平平淡淡的幸福，还有人追求富贵奢华的生活。这些不都是美好的理想吗？或许有一天，他们过上了曾经梦想的生活，那么在通往梦想的这条路上，他们已经到达了终点。一路上，有阳光的照耀，亦有风雨的陪伴，他们该是不孤单的吧？或许有的人还在路上艰难的行走着，他们该是充满力量的吧？又或许这条路根本没有尽
头，但路上的风景确实是美丽的吧？ 我也有梦想，而且我有很多梦想。 小时候我喜欢看电视，每当看到英雄侠客的出现，顿时肃然起敬，好想成为他们那样的人，可以无拘无束，浪迹天涯，该是多么自在啊！每当看到那些英姿飒爽的士兵在战场上冲锋陷阵的身影，就好渴望将来能够像他们一样保家卫国，做国家的坚强捍卫者。每当看到那些威武严肃的警察，又会以他们为榜样，憧憬未来如他们一般一身正气，为社会惩恶扬善，向人们弘扬正义。那时的想法真的好单纯，只是想想，就会觉得幸福。 后来我上小学了，认识了很多小朋友，我们一起玩耍，一起做作业，他们陪伴我度过了快乐的童年。但唯一令我遗憾的是，父母没能看着我一点点长大。因为农村人唯一的生活来源就是那点微薄的耕地收入，然而家里开销大，经常入不敷出，所以父母只好去外地打工挣钱来填补家用。至此，父母踏上了漫漫的打工之旅。那时的我还不明白父母背着包裹要干嘛，直到思念的滋味冒上心头，我才开始慌张，才哭着着急寻找他们。为此，爷爷奶奶花了好多时间来哄我，他们对我说，爸妈是出去挣钱了，只要你乖乖的，他们就会很快回来，并且会给你买好多礼物。我渐渐接受他们的说法，不嚷嚷着找他们了，但心里却更渴望见到他们，想着他们回来时带的礼物是什么。就这样盼着盼着，终于要过年了，父母该回来了，那段时间的我就会特别高兴。而每次爸妈归来时，都给我买了好多吃的和玩的礼物，有时还有漂亮的衣裳，看着爸妈脸上的笑容，我也幸福地笑了。所以，在小学的时
大学生为中国梦可以做什么
青年大学生如何为实现中国梦作出贡献 大学生是“中国梦”的寄托者，“中国梦”的本质内涵是实现国家富强、民族复兴、人民幸福，而青年学生是祖国的未来、民族的希望，是党和人民事业发展朝气蓬勃的推动力量。我们中 国人，几千年来，做了三个梦：天下梦，国家梦，个人梦。天下梦就是大同梦，国家梦就是强 国梦，个人梦就是幸福梦。这些梦是互为前提，彼此成全的。中国的道路，不是理论问题，是 实践问题。这是易中天教授在北大的演讲中所提到的中国梦！现在大家都在讨论中国梦。我 认为，实现中华民族伟大复兴，就是中华民族近代以来最伟大的梦想。这个梦想凝聚了几代 中国人的夙愿，体现了中华民族和中国人民的整体利益，是每一个中华儿女的共同期盼。当 代大学生承担历史的重任，是社会上富有朝气、充满生命力的群体。良好的形象不仅是大学生 成才的一个重要方面，也是社会对大学生的要求。同学们要适应时代要求，自觉地塑造积极健 康向上的崭新形象。所以，必须要做到以下几点：第一，大学生应培养社会主义核心价值观。核心价值观是一个社会中居统治地位、起支配作用的核心理念，也是一个社会必须长期普遍 遵循的基本价值准则，具有相对稳定的特点。社会主义价值观是对社会主义价值的总的看法 和最根本观点。在党的十七大召开之前的十六届六中全会上，党第一次提出了社会主义核心 价值体系这一科学命题,并对社会主义核心价值体系的科学内涵作了界定。社会主义核心价值 体系包括四个方面的基本内容，即马克思主义指导思想、中国特色社会主义共同理想、以爱 国主义为核心的民族精神和以改革创新为核心的时代精神、以“八荣八耻”为主要内容的社会主 义荣辱观。大学生正处于人生观、价值观形成的关键时期，他们思想观念逐渐趋于成型，但仍 具有较大的可塑性；他们接受新鲜事物的能力很强，但鉴别力明显欠缺。赢得青年就赢得未来，我们以社会主义核心价值观加强大学生思想教育，具有鲜明的时代意义和现实意义。第二， 当代大学生应该努力学习勤奋刻苦，善于合理利用学习时间，在学习中起表率作用。这是我 作为一名学生党员首先应该做到的，在努力让自己做到优秀的同时，和大家共同进步，经常 交流学习经验，不保留，乐于帮助后进的同学。另外，提高自己在知识的摄取方面的宽度和 深度，在学好专业知识的同时还要注意个人文化修养的培养，增加自己的知识面，丰富自己 的文化底蕴。与此同时，也应该学好理论，提高素质，在政治上带动同学进步。第三，增强 党性修养，增强党性修养。要有坚定的马克思主义信仰，要讲政治道德，要自觉提高政治道 德修养。要讲大局，在认真学习和积极宣传党的先进思想、政治理论的基础上，要从实际出发、发挥带头作用，勇于实践“三个代表”重要思想，积极参加社会实践，在实践中检验所学 的理论知识，并不断提高自己的思想修养和理论水平。 第四，追求真理、善于创新。当代大学生应当发挥朝气蓬勃、思维敏捷、敢为人先、最少 陈旧观念、最多创造活力的诸多优势，坚持追求真理的精神，不断夯实科学文化知识基础，掌 握善于创新的技能，努力提高持续创新能力，使自己成为祖国和人民需要的、富有创新精神 的高素质人才。要善于从马克思主义理论中汲取营养，树立科学的世界观，把握正确的方法论，努力做科学探索和创新的先锋。第五、德才兼备、全面发展。当代大学生要掌握扎实的专业 基础知识和最前沿的科学文化知识，以造福国家人民。没有坚实的科学知识，就不能发展经济，更谈不上建设社会主义现代化。同时，要坚持以德为先，德才兼备。中目前社会上出现的社会 腐败和高科技犯罪等现象，为人们敲响了正确把握德才关系的警钟。对当代大学生来说，“德” 绝不是可有可无的。德才兼备是衡量大学生全面发展的一个重要标准。第六、知行统一、脚 踏实地。当代大学生要努力将书本知识和实际行动密切联系起来，塑造“知行统一、脚踏实地” 的良好形象。“知行统一”式和道德人格紧密结合在一起的。一个人能否做到言行一致，是他能否在立身处世等方面取得成功的重要条件。我们在日常的学习和生活中，要时时提醒自己，比 如应该做的事情，认识到了，但是是否做到了；应该改正的错误，认识到了，但是否改正了。一个大学生如果能够从身边的事情做起，做到言行一致，老老实实做人，踏踏实实做事，他的 道德人格必然会不断完善。我们应该从现在做起，从我作起，在平时生活中严格按照共产党 员的标准要求自己，多关注国家大事，刻苦学习，努力奋斗，争取成为国家的有用之才，将 来为祖国的社会主义现代化建设作出自己应有的贡献，为全面建成小康社会，实现中华民族的 伟大复兴贡献积极力量。