Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special^® enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N^® enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.     

Hydrolysis of rapeseed protein isolates: Kinetics,characterization and functional properties of hydrolysates

Two rapeseed protein isolates corresponding to albumins and globulins, respectively, were produced from an industrial defatted rapeseed meal. A pilot-scale process of protein extraction has been developed to remove major anti-nutritional compounds, easy to scale-up and using recyclable solvents. The kinetics of the hydrolyses of these two protein substrates using Alcalase 2.4L^® were compared by the measurement of the degree of hydrolysis (DH) when varying the initial proteins and enzyme concentrations. The globulins isolate was hydrolysed more efficiently than the albumins isolate mainly due to the compact and globular conformation of albumin (napin). Kinetic parameters have been determined for both substrates and a log-linear relation has been established between the DH values at a definite time and the initial enzyme/substrate ratio. Such relationships allow an effective monitoring of hydrolysis process since the hydrolysates analysis using reverse-phase chromatography coupled with mass spectrometry revealed that peptide maps corresponding to peptides of molecular weight inferior to 1 kDa are similar at a specific DH, independently of the reaction temperature and initial concentrations of substrate and enzyme. Thus, it is demonstrated that the DH is the sole parameter needed to control the physico-chemical properties and consequently the functionalities (solubility, foaming and emulsifying properties) which depend on the nature of peptides present in the hydrolysates.     

蛋白水解物制备工艺及其在生物技术领域中的应用研究进展

蛋白水解物是蛋白质经酶、酸、碱和发酵等方法水解得到的混合物,由于其含有丰富的氨基酸和多肽,并为细胞生长提供多种营养成分、贴壁因子及生长因子类似物等,从而在生物技术领域中得到了广泛的应用。本文综述了蛋白水解物制备工艺、在生物技术领域中的应用及其在细胞培养中对细胞增殖、代谢的影响,以期为蛋白水解物的开发和应用提供参考。     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.     

Development of an animal‐component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h^?1 were achieved in IPT‐AFM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Secretagogues and Growth Factors in Fish and Crustacean Protein Hydrolysates

The search for new molecules in fish protein hydrolysates is of great interest in animal feeding as it is in aquaculture, fertilizer, cosmetic, and pharmacologic domains. Different sources of hydrolysates such as shrimp waste (Pandalus borealis), cod (Gadus morhua) head, and head and viscera of sardine (Sardina pilchardus), obtained after hydrolysis or autolysis, were tested on fibroblast cell cultures and by gastrin radioimmunoassay. The level of hydrolysis seems to play an important role in the presence of biological peptides. Elution profile on a gel filtration Sephadex G-50 column was used to estimate the degree of hydrolysis of the fractions studied. Growth-factor-like activities were found in less-hydrolyzed fractions. Conversely, the most-hydrolyzed fractions showed gastrin and cholecystokinin immunoreactivity. Received January 22, 1999; accepted April 13, 1999     

The role of iron in the growth of human leukemic cell lines

The growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum-free medium, the cells require insulin, transferrin, and albumin. Two highly water-soluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen-stimulated lymphocytes were grown in serum-free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free-iron salts are taken up by the cells by a mechanism that is completely independent from transferrin-receptors.     

A serum-free medium for hybridoma cell culture

The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA bovine serum albumin - CS calf serum - DMEM Dulbecco's modified Eagle's medium - ELISA enzyme-linked immunosorbant assay - McAb monoclonal antibody - PEG polyethylene glycol - SFM serum-free medium     

Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10⁶ cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10⁶ cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10⁶cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

Many studies underlined the great benefits of hydrolysates used as additives in animal free media on cell culture performances. However, to precisely define hydrolysate supplementation strategies, a deeper understanding of their effect on cell growth and protein production is required. In the present study, the effect of addition of one yeast extract (YE) and two yeast peptones (named YP.A and YP.B) in a chemically defined medium was first assessed on cell culture performances. Interestingly, specific effects were found depending on the degree of degradation of yeast hydrolysates. The YE at 1 g L⁻¹ increased the maximal cell density by 70 %, while a mixture of YE (1 g L⁻¹) and YP.A (4 g L⁻¹) increased IgG production by 180 %. These conditions were then evaluated on the CHO cell kinetics all over cultures. Hydrolysates extended the cell growth phase in Erlenmeyer flask and increased the maximal growth rate in bioreactor up to 20 %. Cell growth stimulation induced by hydrolysates addition was linked with energetic metabolism improvement suggesting that they promote oxidative pathway. Furthermore, hydrolysates provided an additional source of substrate that supported cell growth despite glutamine limitation.     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Growth of Im-9 Human Lymphoblasts In Serum-Free Medium: Stimulation By Glucocorticoids

Abstract. The IM-9 human B-lymphoblast cell line grows well in a completely defined serum-free medium containing insulin, transferrin, low density lipoprotein and oleic acid in complex with fatty acid-free bovine serum albumin. Growth of the IM-9 cells is stimulated by addition of physiological concentrations of hydrocortisone to this medium. the order of growth stimulatory potency of several steroids is dexamethasone > hydrocortisone > aldosterone, whereas testosterone does not stimulate growth of the IM-9 cells. This order of potency suggests that the effect is mediated by binding to glucocorticoid receptors. Growth of the IM-9 cells is also stimulated by the neuropeptide substance P. the defined serum-free medium described in this report will be useful for further studies of the biological responses of the IM-9 cells to other hormones in the absence of interference from hormones and growth factors present in serum.     

Growth of IM-9 human lymphoblasts in serum-free medium: stimulation by glucocorticoids

The IM-9 human B-lymphoblast cell line grows well in a completely defined serum-free medium containing insulin, transferrin, low density lipoprotein and oleic acid in complex with fatty acid-free bovine serum albumin. Growth of the IM-9 cells is stimulated by addition of physiological concentrations of hydrocortisone to this medium. The order of growth stimulatory potency of several steroids is dexamethasone greater than hydrocortisone greater than aldosterone, whereas testosterone does not stimulate growth of the IM-9 cells. This order of potency suggests that the effect is mediated by binding to glucocorticoid receptors. Growth of the IM-9 cells is also stimulated by the neuropeptide substance P. The defined serum-free medium described in this report will be useful for further studies of the biological responses of the IM-9 cells to other hormones in the absence of interference from hormones and growth factors present in serum.     

Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture,clonal growth in low density culture,and stimulation to enter S phase in resting culture

Summary A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Chemometric analysis of soy protein hydrolysates used in animal cell culture for IgG production – An untargeted metabolomics approach

Soy protein hydrolysates are used as the most cost effective medium supplement to enhance cell growth and recombinant protein productivity in cell cultures. Such hydrolysates contain diverse classes of compounds, such as peptides, carbohydrates and phenolic compounds. To identify if specific compounds dominate the functionality of hydrolysates in cell cultures, thirty samples of hydrolysates with different cell culture performances were analyzed for chemical composition using an untargeted metabolomics approach. Out of 410 detected compounds, 157 were annotated. Most of the remaining 253 compounds were identified as peptides, but could not be annotated exactly. All compounds were quantified relatively, based on their average signal intensities. The cell growth and total immunoglobulin (IgG) production, relative to the CD medium (100%), ranged from 148 to 438% and 117 to 283%, respectively. Using bootstrapped stepwise regression (BSR), the compounds with the highest inclusion frequency were identified. The most important compound, i.e. phenyllactate and ferulate explained 29% and 30% of the variance for cell growth and total IgG production, respectively. Surprisingly, all compounds identified in the BSR showed a positive correlation with cell growth and total IgG production. This knowledge can be applied to monitor the production and accumulation of these compounds during the production process of hydrolysates. Consequently, the processing conditions can be modulated to produce soy protein hydrolysates with enhanced and consistent cell culture performance.     

Basic fibroblast growth factor,albumin, and transferrin purified from rat rhodamine fibrosarcoma tissue are all essential for growth of primary tumor cells from the same tissue in serum-free medium

Summary Primary Rhodamine fibrosarcoma (RdF) cells from rats were shown to grow in serum-free medium supplemented with basic fibroblast growth factor (bFGF), albumin, and transferrin, all of which were purified from RdF tissue. Their growth rate with these supplements was similar to that of cells in medium supplemented with calf serum. bFGF purified from RdF tissue (Rd-bFGF), which was previously designated as DNA synthesis factor, stimulated the growth of primary RdF cells maximally at 30 ng/ml in the presence of the other two proteins. Albumin and transferrin were separated from partially purified tumor growth stimulating activity which was previously shown to stimulate growth of primary RdF cells in serum-free medium. The albumin (RdA) and transferrin (RdT) found in the extract of RdF tissue were not due simply to contamination of the tissue with blood, but to their accumulations in the tissue. The growth stimulatory activities of RdA and RdT on primary RdF cells in serum-free medium were maximal at 30 and 10 μg/ml, respectively. These results suggest that Rd-bFGF, RdA, and RdT, all of which accumulate in the tumor tissue, are essential for growth of RdF cells in the tissue. This work was supported by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture of Japan.     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.