Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 μM CuSO₄ where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.     

Imbalance of morphofunctional responses of Jurkat T lymphoblasts at short-term culturing with relief zinc- or copper-containing calcium phosphate coating on titanium

Morphofunctional response of Jurkat T cells that were cultured for 24 h on substrates prepared from commercially pure titanium with relief microarc bilateral calcium phosphate coating containing copper or zinc was studied. Changes in the concentration of essential trace elements contained in this coating can cause significant imbalance of molecular processes of differentiation, secretion, apoptosis, and necrosis and reduce tumor cell survival.     

Effect of the Copper Content of Distillery Spent Wash on its Utilization for Single-cell Protein Production by Geotrichum candidum, Hansenula anomala and Candida kruzei

Geotrichum candidum, Hansenula anomala and Candida kruzei were grown in batch and continuous culture on samples of the spent wash from a malt whiskey distillery containing levels of dissolved copper up to 22 mg/l. In batch culture some inhibition of growth at the higher copper concentrations and as much as a sixfold accumulation of copper in the biomass were observed. In Geotrichum candidum this accumulation occurred in three distinct phases during the period of batch growth. Although biomass yields in mixed continuous culture declined with increasing dilution rate there was a corresponding increase in cell copper concentration such that total copper removal from the medium remained approximately constant.     

单克隆抗体Mab-A生产细胞Sp2/0缩小模型培养优化研究

Sp2/0是一种生产单克隆抗体的常用细胞株。本研究首先在批次培养模式中对适合Sp2/0细胞生长的5种基础培养基、摇床转速、培养温度、二氧化碳浓度、微量元素和GlutaMAX ^TM替换谷氨酰胺等影响因素进行了筛选研究。结果显示Sp2/0细胞在批次培养中细胞密度最高值达到13.12×10 ⁶ cells/ml,培养时间为7天。除培养温度会导致不同的细胞生长密度和活率、进而影响培养时间外,其它因素不能导致明显的细胞生长差异。随后在流加培养模式下就14种补料组合进行了筛选,Sp2/0在流加培养模式下细胞的峰值密度可达20~30×10 ⁶ cells/ml,培养时间9天,单克隆抗体Mab-A日产量最高达到27.20mg/L。最后应用批次-反复流加培养模式培养Sp2/0细胞,该条件下峰值细胞数为50.42×10 ⁶ cells/ml,培养时间14天,每天单抗产量(141.10mg/L)是流加培养的5.19倍。这些研究结果为Sp2/0细胞规模化生产单克隆抗体奠定了一定基础。     

ZINC AND COPPER TOLERANCE OF AGROSTIS STOLONIFERA L. IN TISSUE CULTURE

Callus tissue was induced from shoot meristematic tissue and root tips of a clone of the grass Agrostis stolonifera tolerant to both zinc and copper, and from a control clone tolerant to neither metal. Growth of the callus tissue on media containing zinc and copper showed that tolerance to both metals was maintained in tissue culture. The pattern of metal uptake in tissue culture resembled uptake by whole plants in that tolerant tissue took up more metal than nontolerant tissue. Plants regenerated from callus had the same copper and zinc tolerance as the original parental clones regardless of time of growth in tissue culture and shoot or root origin of the tissue. The results support previous evidence that metal tolerance is genetically determined and acts at the cellular level.     

Xanthan gum: an economical substitute for agar in plant tissue culture media

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.     

Evaluation of the batch kinetics of the amino acid metabolism of a mouse hybridoma and human lymphoblastoid cell line using a pre-column FDNDEA derivitisation,HPLC technique

Summary The analysis of free amino acids in mammalian cell culture media can give valuable information on the metabolism of particular commercially valuable cell lines. Analysis of spent culture media indicates potential limiting nutrients. However, analysis over the whole culture period enables a kinetic approach to allow understanding of amino acid uptake and potential strategies to improve media design. This communication details the use of a less commonly used HPLC protocol, indicating various advantages and disadvantages. Further, the batch kinetics of amino acid metabolism of two cell lines are discussed.     

Observations on the use of a cation exchange resin for the preparation of metal-depleted media for lymphocyte culture

A chelating resin specific for divalent cations (Chelex) was used to prepare metal-depleted media for lymphocyte culture. A batch procedure (resin in pH 7.4 phosphate buffer/specimen, 1:1) removed 70-80% of iron, 77-87% of copper and 88-98% of zinc, calcium and magnesium. At variance with other reports, when a resin/specimen ratio of 1:4 was used, iron chelation decreased to 40%, whereas other cation chelation remained unchanged. Best chelation for iron and calcium was obtained at pH 5-6.4; for copper, zinc and magnesium, at pH 7.4-8.0. During the procedure protein content decreased by 8-10%; arginine and lysine by 80%; asparagine, cystine, tyrosine and phenylalanine by 60%, other amino acids by 35%. These new data suggest that cation-depleted media prepared with Chelex may be used to study the effects of cations on lymphocytes in culture, provided that the most appropriate pH and resin/specimen ratio are selected and adequate amino acid replacement is performed. Results on normal human lymphocytes are reported.     

Copper deficiency in potato dextrose agar causes reduced pigmentation in cultures of various fungi

Potato dextrose agar (PDA) is one of the most commonly used media for the isolation and cultivation of fungi, with morphological features and pigmentation in culture often being important for identification of cultures. Cultivation of various fungi on different brands and batches of powdered (commercial) potato dextrose media revealed deficient pigmentation in five of 10 media tested. Reduced pigmentation on these media was correlated with low levels of copper and colony colour was restored by the addition of copper. Deficient pigmentation was most pronounced when copper levels in the medium were below 50 ng mL(-1) (50 p.p.b.; 0.8 microM). Differences in pigmentation and laccase activity of spore and mycelial preparations were quantified for representative species belonging to the genera Aspergillus, Fusarium, Trichoderma, Cladosporium and Penicillium grown on PDA containing different amounts of copper. A strong positive correlation between laccase activity and copper levels was observed. Differences were also found between batches of raw potatoes, with organically cultivated tubers having higher copper levels than those grown by conventional methods, possibly because of the use of copper-based fungicides in the former case. Routine addition of 1000 ng mL(-1) copper (or standard trace element solutions) to PDA and other undefined media is advised to avoid atypical culture pigmentation and possibly other consequences of reduced activity of copper-requiring enzymes.     

Copper and iron concentrations in Ascophyllum nodosum (Fucales, Phaeophyta) from different sites in Ireland and after culture experiments in relation to thallus age and epiphytism

In laboratory experiments, copper concentrations in plants of Ascophyllum nodosum (L.) Le Jolis (Fucales, Phaeophyta) increased with the concentrations in the culture media and were highest in younger, meristematic thallus parts. After initial accumulation in high-copper medium and subsequent transfer to clean seawater for 5 days, no release of copper could be detected. Iron concentrations in A. nodosum tissue were not related to concentrations in the culture medium. Differences between copper concentrations in plants from different sites in areas with high yachting activity in Strangford Lough, Northern Ireland, could be explained by differences in water motion and human activity, in particular the application of copper-releasing antifouling paints to leisure boats. Iron concentrations were also highest in plants from the sheltered, polluted site but did not differ significantly between the other two sites. No differences in copper nor iron concentrations were found between different-aged thallus parts of plants from any site. X-ray microanalysis revealed that most of the iron detected was located in epiphytic pennate diatoms on the A. nodosum surface. In thallus areas without diatoms, iron levels were below the detection limit for X-ray microanalysis. Mapping for copper indicated that most of the accumulated copper was located in cells near and immediately below the thallus surface. "Epidermis"-shedding occurred in plants from the culture experiments and also in freshly-collected material and may have resulted in a loss of metal ions accumulated by surface cells and by epiphytic diatoms. The results suggest that A. nodosum could be used as a biological indicator for copper but not for iron, and that young, apical plant parts are most sensitive to changes in metal concentrations in the water.     

Biosorption of lead and copper from aqueous solutions by pre-treated crab and arca shell biomass

Sorption potential of pretreated crab and arca shell biomass for lead and copper from aqueous media was explored. The effects of pH, initial concentration, biosorbent dosage and contact time were studied in batch experiments. Effects of common ions like sodium, potassium, calcium and magnesium on the sorption capacity of pretreated crab and arca biomasses were also studied. At equilibrium, the maximum uptake by crab shell biomass was 19.83+/-0.29 and 38.62+/-1.27 mg/g for lead and copper, respectively. In case of arca shell biomass the maximum uptake capacity was 18.33+/-0.44 mg/g and 17.64+/-0.31 mg/g for lead and copper, respectively. Combined effect of all the common ions up to 50 microg/ml concentration was negligible for both the metals using both biomasses. Sorption isotherms were studied to explain the removal mechanism of both elements by fitting isotherms data into Lagergren, Freundlich and Langmuir equations.     

Bioreactor productivity and media cost comparison for different intensified cell culture processes

Process intensification in biomanufacturing has attracted a great deal of interest in recent years. Manufacturing platform improvements leading to higher cell density and bioreactor productivity have been pursued. Here we evaluated a variety of intensified mammalian cell culture processes for producing monoclonal antibodies. Cell culture operational modes including fed‐batch (normal seeding density or high seeding density with N‐1 perfusion), perfusion, and concentrated fed‐batch (CFB) were assessed using the same media set with the same Chinese Hamster Ovary (CHO) cell line. Limited media modification was done to quickly fit the media set to different operational modes. Perfusion and CFB processes were developed using an alternating tangential flow filtration device. Independent of the operational modes, comparable cell specific productivity (fed‐batch: 29.4 pg/cell/day; fed‐batch with N‐1 perfusion: 32.0 pg/cell/day; perfusion: 31.0 pg/cell/day; CFB: 20.1 – 45.1 pg/cell/day) was reached with similar media conditions. Continuous media exchange enabled much higher bioreactor productivity in the perfusion (up to 2.29 g/L/day) and CFB processes (up to 2.04 g/L/day), compared with that in the fed‐batch processes (ranging from 0.39 to 0.49 g/L/day), largely due to the higher cell density maintained. Furthermore, media cost per gram of antibody produced from perfusion was found to be highly comparable with that from fed‐batch; and the media cost for CFB was the highest due to the short batch duration. Our experimental data supports the argument that media cost for perfusion process could be even lower than that in a fed‐batch process, as long as sufficient bioreactor productivity is achieved. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:867–878, 2017     

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.     

Measurement of trace elements in murine liver tissue samples: Comparison between ICP-MS/MS and TXRF

BackgroundTrace elements exhibit essential functions in many physiological processes. Thus, for research focusing on trace element homeostasis and metabolism analytical methods allowing for multi-element analyses are fundamental. Small sample amounts may be a big challenge in trace element analyses especially if also other end points want to be addressed in the same sample. Therefore, the aim of the present study was to examine trace elements (iron, copper, zinc, and selenium) in murine liver tissue prepared by a RIPA buffer-based lyses method.Methods and resultsAfter centrifugation, lysates and pellets were obtained and trace elements were analyzed with TXRF in liver lysates. The results were compared to that obtained by a standard microwave-assisted acidic digestion with subsequent ICP-MS/MS analysis of the same liver tissue, liver lysates, and remaining pellets. In addition, trace element concentrations, determined in murine serum with both methods, were compared. For serum samples, both TXRF and ICP-MS/MS provide similar and highly correlating results. Furthermore, in liver lysate samples prepared with RIPA buffer, comparable trace element concentrations were measured by TXRF as with the standard digestion technique and ICP-MS/MS. Only marginal amounts of trace elements were detected in the pellets.ConclusionTaken together, the results obtained by the present study indicate that the RIPA buffer-based method is suitable for sample preparation for trace element analyses via TXRF, at least for the here investigated murine liver samples.     

The role of catecholestrogens in placental steroidogenesis

The effect of the catecholestrogen, 2-hydroxyestrone (2-OHE1), on placental steroidogenesis was studied by incubating 2-OHE1 with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHE1 stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the alpha-adrenergic blocker, phenoxybenzamine, and the beta-adrenergic blocker, propranolol. We conclude that 2-OHE1 affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors.     

Needless Retesting of Quality-Assured, Commercially Prepared Culture Media

Current revival of interest in quality control of bacteriological culture media prompted this laboratory to develop and implement a system of quality control testing of media purchased from commercial manufacturers. During 8 months we tested more than 900 lots of 46 different media representing 350,000 units of culture media purchased from two major and one minor supplier(s). Only 17 lots were found to be unsatisfactory. This experience raised a question about the real necessity for extensive retesting by users of commercially prepared, quality-assured media. It is suggested that primary responsibility for quality control be placed on the relatively few manufacturer-vendors rather than on the multiple purchaser-users, who may not possess either the expertise or the resources for quality control programs.     

Development and manufacturability assessment of chemically‐defined medium for the production of protein therapeutics in CHO cells

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     

Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Neurite-promoting activities for embryonic spinal neurons and their developmental changes in the chick

Spinal motoneurons may depend upon muscle-derived factors for axon outgrowth and stabilization at two principal stages of their development: during the initial invasion of the differentiating muscle masses in the embryo and during the perinatal regression of multiple innervation. Using a bioassay involving the measurement of neurite outgrowth from 4.5-day embryonic chick spinal neurons in dissociated cell culture, neurite-promoting activities were detected both in medium conditioned over embryonic chicken myotubes in vitro (embryonic muscle-conditioned medium) and in soluble extracts of chick leg muscle prepared 3-5 days after hatching (postnatal muscle extract). The molecules responsible for these two activities had physicochemical properties that distinguished them both from each other and from some other reported neurite-promoting factors. The factor in embryonic muscle-conditioned medium, although active on uncoated tissue culture wells, bound with only low affinity to tissue culture plastic under cell culture conditions. It was inactivated by incubation with trypsin, and was essentially found only in media conditioned by muscle and liver cells. The factor in PNME, on the other hand, bound to plastic culture wells and was found in extracts of a variety of tissues. Its concentration in postnatal leg muscle was developmentally regulated: the specific activity increased approximately 10-fold between hatching and Day 3 (maximum value: 3200 units/mg protein) and then fell back to nearly its original levels by Day 7. Evidence is presented that the observed effects of these two neurite-promoting factors did not result from differential survival in vitro of different cell subpopulations. Possible roles for the two active factors during motoneuron development are discussed.     

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue in vitro

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.