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1.
Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA
Analysis of variance
- BSA
bovine serum albumin
- CHO
Chinese hamster ovary
- FBS
fetal bovine serum
- MTT
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
- PBS
phosphate buffered saline
- SFM
serum-free medium 相似文献
2.
The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown. 相似文献
3.
研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。 相似文献
4.
Chen Zhaolie Xiao Chengzu Liu Hong Wu Benchuan Jia Xihua 《Biotechnology Techniques》1996,10(6):449-452
Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l06cells/ml. 相似文献
5.
Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can
be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium.
Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of
viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation
(basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although
they do not fully substitute for BPE. 相似文献
6.
François LeFloch Bertrand Tessier Sébastien Chenuet Jean-Marc Guillaume Pierre Cans Jean-Louis Goergen Annie Marc 《Cytotechnology》2006,52(1):39-53
The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure
of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product,
particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent
on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to
serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO
cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes
as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when
cells were adapted to SFX-CHOTM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in
the enriched SFMTM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the
serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release
a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell
adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of
protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells. 相似文献
7.
Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium 总被引:1,自引:0,他引:1
Sébastien Quesney Annie Marc Catherine Gerdil Cyrille Gimenez Jacqueline Marvel Yves Richard Bernard Meignier 《Cytotechnology》2003,42(1):1-11
The aim of this study was to understand the metabolism kinetics of Vero cells grown on microcarriers in bioreactors in serum-free
medium (SFM). We sought to determine what nutrients are essential for Vero cells and how they are consumed. Contrary to glucose
and to most of the amino acids, glutamine and serine were very quickly depleted in this medium and can be supposed to be responsible
for cell apoptosis. Lactate and ammonium ions did not reach toxic levels for Vero cells. We payed more attention to the lactate
metabolism. Usually we observed that after about 2 days lactate was consumed in serum-containing media, but its concentration
plateaud in SFM. Moreover, the addition of serum in SFM provoked lactate consumption and the rate of glucose and glutamine
consumption was twice as high as in the SFM not supplemented with serum. The depletion of glutamine and serine and the metabolic
deviations leading to a shortage of intermediate products required for other metabolic pathways probably contribute to the
lower cell yield and higher cell death rate in SFM.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
8.
Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells
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Wai Lam W. Ling Yunling Bai Cheng Cheng Ishai Padawer Changjian Wu 《Biotechnology progress》2015,31(5):1163-1171
Advantages of using internally developed chemically‐defined (CD) media for cell culture‐based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in‐house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large‐scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large‐scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb‐expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1163–1171, 2015 相似文献
9.
The addition of 2.8 g/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10–4 vs. 2.48×10–4 IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium). 相似文献
10.
中华仓鼠卵巢(CHO)工程细胞无血清培养的研究 总被引:2,自引:0,他引:2
以DMEM:F12(1:1)为基础培养基,通过观察细胞生长状态和检测乙肝表面抗原的表达量作为评价指标,筛选适合于CHO工程细胞生长的生长因子,如:胰岛素、转铁蛋白、氢化可的松、硒酸钠,丁二胺等。并且建立了J5SFM培养基。该培养基与商品化的无血清培养基比较,能够使细胞生长维持较长的时间,表达产物分泌量也相对较高。 相似文献
11.
Development of a serum-free medium for the production of humanized antibody from chinese hamster ovary cells using a statistical design 总被引:1,自引:0,他引:1
Eun Jung Kim No Soo Kim Gyun Min Lee 《In vitro cellular & developmental biology. Animal》1998,34(10):757-761
Summary To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO) cells,
a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing
α-minimal essential medium (α-MEM) with Fe(NO3)3·9H2O, CuCl2, ZnSO4·7H2O, and Na2SeO3 which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to
the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine
were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown
to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium
with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in
this SF medium were comparable to those in α-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results
obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing
a humanized antibody. 相似文献
12.
Hubert Schwarz Kevin Lee Andreas Castan Veronique Chotteau 《Biotechnology and bioengineering》2023,120(9):2523-2541
A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 106 and 120 × 106 cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry. 相似文献
13.
Adle Martial Isabelle Gaillard Jean-Marc Engasser Annie Marc 《Enzyme and microbial technology》1995,17(12):1062-1066
A homemade serum-free medium containing a low protein level under 0.1 g l−1 has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid,
- dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l−1. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l−1 of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture. 相似文献
14.
Jun-ichiro Hata Takeyoshi Tamura Soji Yokoshima Shinya Yamashita Shoko Kabeno Ken Matsumoto Kazukiyo Onodera 《Cytotechnology》1992,10(1):9-14
A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO
Chinese hamster ovary
- hGH
human growth hormone
- dFCS
dialyzed fetal calf serum
- dhfr
dihydroforate reductase
- MTX
methotrexate 相似文献
15.
Selection and optimization of the concentrations of chemically defined components effective on recombinant factor VIII:C production by CHO cells have been performed with statistically designed experiments. Screening of efficient compounds on factor VIII expression was performed by using HADAMARD method, while a precise optimization of the concentrations of two components was achieved with the help of DOEHLERT method. Increased specific activity of factor VIII and reduced cost medium have been obtained with a fewer number of experiments than compared to a less rational approach. 相似文献
16.
张香玲 《微生物学免疫学进展》2015,(2):67-72
Vero细胞是世界卫生组织和《中国药典》认可的用于人用疫苗和动物疫苗生产的细胞系,Vero细胞无血清培养生产疫苗已成为当前的主要趋势。无血清培养的关键是设计符合Vero细胞贴壁特性和提高细胞密度的无血清培养基,这也是规模化培养的关键因素之一。Vero细胞无血清培养基的开发与使用一方面减少了对动物血清的依赖,提高了病毒性疫苗的质量安全;另一方面促进了无血清培养技术的发展与应用。现就Vero细胞无血清培养基的研究进展予以综述。 相似文献
17.
Shuichi Hashizume Kazuhiko Kuroda Hiroki Murakami 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,763(4):377-382
A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin. 相似文献
18.
以悬浮适应的表达尿激酶原CHO工程细胞为研究对象,在100mL的摇瓶中进行无血清悬浮培养,以细胞密度、细胞活力、Pro-UK活性、葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、乳酸对葡萄糖的得率系数(Ylac/glc)为观察指标,同时以细胞有血清悬浮培养作为参照,考察CHO工程细胞无血清悬浮培养生长和代谢特征。观察结果表明,CHO工程细胞在无血清及有血清悬浮培养条件下表现为大致相似的细胞生长和代谢特征。在此基础上,依据实际检测的数据,应用MATLAB软件对细胞对数生长期的细胞生长、乳酸生成及葡萄糖消耗的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了细胞在无血清培养条件下的生长及代谢动力学模型。 相似文献
19.
Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium 总被引:2,自引:0,他引:2
Francois Gasser Philippe Mulsant Michel Gillois 《In vitro cellular & developmental biology. Plant》1985,21(10):588-592
Summary A new synthetic medium (referred to as GC3) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's
F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and
selenium (1×10−7
M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium)
also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading.
Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high
density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium. 相似文献
20.
Fanny Knöspel Rudolf K. Schindler Marc Lübberstedt Stephanie Petzolt Jörg C. Gerlach Katrin Zeilinger 《Cytotechnology》2010,62(6):557-571
The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture
media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells,
the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of
a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis
according to Plackett–Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence
on the proliferation activity of the cells, while zinc and l-cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following
factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays
are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media
for human embryonic stem cells (hESC). 相似文献