Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium.

The effect of polyvinyl formal (PVF) culture surface on the growth of 10 mammalian continuous cell lines, including Swiss 3T6, NCTC clone 929 L, BHK-21 clone 13, CHO-K1, PK 15, A 431, HeLa, MDCK, LLC-MK2 and Vero in protein-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with trace elements and L-ascorbic acid 2-phosphate, was investigated. Most of the cell lines showed only some initial proliferation on PVF similar to the polystyrene (PS) surface of commercially available culture flasks. In contrast, proliferation of monkey kidney cell line Vero was by far greater on PVF than on PS or poly-D-lysine treated culture surface. In addition, Vero cells on PVF could be subcultured in the protein-free medium without any significant decrease of growth rate in successive passages. These results showed that PVF provides a culture surface which selectively promotes continuous growth of Vero cells in protein-free, chemically defined medium.     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro fertilization of bovine oocytes in protein-free culture system

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.     

In vitro fertilization of bovine oocytes in in-vitro protein-free culture system

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1／Bcl-2或Bcl-2／CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1／Bcl-2和Bcl-2／CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.     

Evaluation of a serum extender,SERASUP II,for cell growth and production of poliomyelitis viruses by VERO cells

Summary A serum extender, SERASUP II, was evaluated for its ability to maintain VERO cell cultures at long term, and for its influence on virus production by cells adapted to that medium. SERASUP II allowed a 90% reduction of the serum amount while maintaining a correct long-term growth. The production of Poliovirus strains 1 and 2 is improved in the extender containing medium, while it is strongly decreased for strain 3.     

Properties of ras-amplified recombinant BHK-21 cells in protein-free culture

We compared serum and protein-free cultures ofa ras-amplified recombinant BHK-21 cell line(ras-rBHK-IgG), which hyperproduces a lungcancer specific recombinant human monoclonal antibody. Ras-rBHK-IgG cells were shown to grow well, evenin protein-free medium and to be morphologicallysimilar to cells cultured in serum containing medium. However, the growth rate of ras-rBHK-IgG cellswas considerably slower in protein-free medium, whichresults in a longer maintenance period compared with cells cultured in serum containing medium. In addition, it was found that antibody production in protein-free culture had a ten times higher maximum than cells cultured in serum containing medium. On theother hand, in high density culture, using the hollowfiber bioreactor system, ras-rBHK-IgG cellscould be maintained for a month in protein-freeculture in contrast with serum culture, which onlylasted for half a month. However, the markedincrease of antibody production was not observed. A total amount of about 15 mg of the recombinantantibody, obtained in protein-free culture, was abouttwo times of that obtained in serum culture, and wasshown to be reactive to lung cancer cells in tissue. From these properties in protein-free medium, it isconcluded that protein-free culture of ras-rBHK-IgG cells is suitable for middle scaleproduction of recombinant human monoclonal antibody.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Comparative growth characteristics of vero cells on gas-permeable and conventional supports

We have evaluated the gas-permeable membrane, etched FEP-teflon, and compared it with other cell supports for tissue culture, in particular Falcon plastic and glass. VERO cells grown on FEP-teflon in 5% CO₂, 95% air reach 1.7 times the density/unit surface area as on other cell surfaces. Cells propagated on FEP-teflon tend to form multiple layers and maintain maximum cell density for periods unusually long for VERO. pH regulation of atmospheric pCO₂, to maintain stable medium pH, increases the maximum cell density on Falcon plastic by 20%, and allows growth on FEP-teflon at serum levels 1.0%. Cells on FEP-teflon eventually form multilayers at these low serum levels. Comparison of the cell morphology in confluent sheets on glass and the top layer of multilayers on FEP-teflon show no significant differences in overall morphology. However, the submerged cells in piled-up foci appear denuded of the small microvilli typical of this cell line. The growth characteristics of FEP-teflon appear related to the permeability of this material to oxygen and/or carbon dioxide. We discuss our findings in terms of the influence of pericellular pO₂, pCO₂ and pH on serum requirements and contact inhibition.     

Comparative development and merozoite production of two isolates of Sarcocystis neurona and Sarcocystis falcatula in cultured cells

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Quality control testing of surfaces for mammalian cell culture using cells propagated in a protein-free medium

Mouse NCTC clone 929 L (L-929) cells were propagated continuously for 3 years as monolayers in a protein-free chemically-defined medium. These cells, designated L-929-WS, were used for quality control testing of the surfaces of commercially available cell culture plastic flasks. Differences in attachment and saturation density of L-929-WS cells in a protein-free culture medium were taken to define various levels of quality of the culture vessels tested. The rate of attachment and growth of L-929-WS cells on a surface of a given quality correlated directly with that of human embryonal fibroblasts and embryonal epithelial cells grown in a serum-free medium supplemented with growth factors and hormones. L-929-WS cells propagated continuously in a protein-free medium provide a simple and sensitive assay system for more general quality control testing of surfaces used for the culture of monolayer cells.     

Roles of various growth factors in growth of human osteosarcoma cells which can grow in protein-free medium.

The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.     

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?10⁶ cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH₄ ⁺ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.     

Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I

In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells grown in protein-free medium. It was found that the use of protein-free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein-free cultures. In addition, membrane fouling occurred more rapidly in protein-free medium than in conventional serum-supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross-flow and vortex-flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vortex-flow filtration of conjuction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridomacells appeared to gradually adapt to increasing rotational speed in the vortex-flow filtration module.     

适于无血清贴壁培养的抗凋亡宿主细胞系CHO-IVB2的构建

应用无血清培养基培养CHO细胞时,由于没有血清提供各种贴壁因子,细胞以悬浮的方式生长。在实际的大规模细胞培养中,CHO细胞往往以贴壁方式培养,要么贴壁于悬浮的微载体中,要么贴壁于固定的聚酯盘状介质或中空纤维中,而很少直接悬浮于培养基中。在无血清培养基中,Vitronectin单一组分可以促使CHO细胞的贴壁和扩增。通过双表达lgf-1和Bcl-2基因,已经构建了可以在无蛋白培养基IMEM中抗凋亡生长的细胞株CHO-IB3。在此基础上,构建了可以同时表达Igf-1、Vitronectin和Bcl-2三个蛋白的三顺反子表达载体pCI—NII—IVB。将该载体转染于CHO—dhfr^-细胞中,构建了一个细胞株CHO—IVB2。该细胞株可以在无蛋白培养基中抗凋亡生长,适于以贴壁的方式大规模培养,用于大量生产外源目的蛋白。     

The establishment and characterization of mouse L-929 cells in protein-free Eagle's Minimal Essential Medium

The mouse cell line L-929 was established in protein-free Eagle's Minimal Essential Medium. The cells have been 'adapted' to continuous growth in the medium using stepwise reductions in the concentration of fetal bovine serum. The cells designated L-929-WS have now been propagated in protein-free Eagle's Minimal Essential Medium for two years. The population-doubling time was about 37 h. The addition of serum stimulated cell growth only slightly, but the saturation density was significantly increased. Morphological examination, a study of the secretion of colony stimulating activity and cytochemical investigations for acid phosphatase and alkaline phosphatase showed that L-929-WS cells, grown in protein-free Eagle's Minimal Essential Medium, did not differ markedly from cells propagated in medium containing serum. The cells provided a simple model for the study of cell growth in the absence of serum or the other macromolecular substances usually added to cell cultures. The general application of the cells for purposes in which the addition of serum or growth factors might interfere, is suggested.