Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Adaptation of mammalian cells to growth in serum-free media

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans     

The growth of Vero cells in suspension as cell-aggregates in serum-free media

Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 10⁶ cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used.     

Erythropoietic progenitor cells from premature neonates studied using a validated serum-deprived medium

We have examined a serum-deprived culture system in order to verify that it is suitable for the study of burst forming unit erythroid (BFU-E) progenitor cells from premature neonates. Optimum growth of BFU-E from premature neonates was observed with each media constituent using the same concentration as that previously described for adult subjects. Growth of immature BFU-E from premature neonates were highly dependant upon a source of Burst Promoting Activity and mature BFU-E derived colonies emerged at day 12 compared to day 14 in adults. Our preliminary results with the validated medium suggest that premature infants have increased peripheral blood concentrations of BFU-E compared to healthy adult controls.Abbreviations Ad Adherent cells - BPA Burst promoting activity - BFU-E Burst forming unit erythroid - Epo Erythropoietin - IL3 Interleukin-3 - LDC Low density (<1.077 g ml¹) peripheral blood mononuclear cells     

Hormonal growth control of cells in culture

Summary Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH₃, can grow in serum-free medium if the medium is supplemented with 3,3′,5-triiodothyronine, TSH-releasing hormone, transferrin, parathyroid hormone, insulin and three isoelectric focusing fractions of blood meal. The blood-meal components can be replaced by fibroblast growth factor and somatomedin C. The growth rate of GH₃ cells in hormone-supplemented serum-free medium is equal to that in serum-supplemented medium, and subculture in such medium is also possible. These results indicate that the replacement of the serum component is complete in the GH₃ system. The hormonal requirements of GH₃ cells and those of HeLa and mouse melanoma, M2R, were compared. Two generalizations could be made: (a) All three cell lines require insulin and transferrin. (b) There is a requirement for a hormone which localizes in the nucleus for each cell line. These generalizations seem to hold true for most of the other cell lines for which the hormonal requirements have been partially worked out. Since insulin is one of the universally required hormones, its effects on GH₃, HeLa and M2R were compared. Insulin stimulates glycogen synthesis in all three cell lines and facilitates fatty-acid synthesis in GH₃ and M2R. However, there is a difference in the effect of insulin on growth among the three cell lines. Insulin is an absolute requirement for GH₃ cells without which the cells cannot survive, whereas this is not the case for HeLa and M2R. The most stringent requirement for HeLa cells is for hydrocortisone, and for M2R, it is for transferrin. These results indicate that even though the necessity for some hormones is common, the degree of requirement may vary from one cell line to another. Whether this difference reflects the difference in the primary mode of action of the hormone on each cell type needs further investigation. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by NIH Grant GM 17019. J. Larner was supported by Josiah Macy Foundation     

SSGF-I,a potent growth-promoting substance for mammalian cells from swine serum

A small amount of swine serum markedly stimulated cell growth for high productivity subclones derived from a mouse human-human heterohybridoma, N12-16.63, secreting an anti-tetanus toxoid human monoclonal antibody in a polyethylene glycol (PEG)-containing serum-free medium, PEG-86-1. A growth promoting substance, SSGF-I, was isolated from the serum by ammonium sulfate fractionation, Cibacron blue F3A-G affinity chromatography, DEAE-agarose ion exchange chromatography, and gel filtrations on Trisacryl GF 2000 and Sephacryl S-300. SSGF-I was characterized as a low density lipoprotein (LDL) of swine serum by its physico-chemical properties. It promoted cell growth synergistically with PEG and its optimum concentration was 1 to 100g/ml. Human LDL was less active, and human or swine high density lipoprotein (HDL) and very low density lipoprotein (VLDL) were inactive. Based on these results, we propose an improved serum-free medium, PEG-86-3, which contains all the ingredients of PEG-86-1 and 10g/ml SSGF-I. This medium is useful for not only high productivity heterohybridomas but also for a variety of lymphoid cell lines.     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells

Microcarriers provide large adhesion area allowing high cell densities in bioreactor systems. This study focused on the investigation of cell adhesion and cell growth characteristics of both anchorage-dependent CHO-K1 and anchorage-independent Ag8 myeloma cell lines cultivated on four different microcarriers (Biosilon®, Microhex®, Cytodex 3®, Cytoline 2®) by considering the cell kinetics and physiological data. Experiments were performed in both static and agitated cell culture systems by using 24-well tissue culture plates and then 50-ml spinner flasks. In agitated cultures, the highest specific growth rates (0.026 h for CHO-K1 and 0.061 h for Ag8 cell line) were obtained with Cytodex 3® and Cytoline 2® microcarriers for CHO-K1 and Ag8 cell line, respectively. Metabolic characteristics showed some variation among the cultures with the four microcarriers. The most significant being the higher production of lactate with microcarriers with CHO-K1 cells relative to the Ag8 cells. SEM analyses revealed the differences in the morphology of the cells along with microcarriers. On Cytodex 3® and Cytoline 2®, CHO-K1 cells attached to the substratum through long, slender filopodia, whereas the cells showed a flat morphology by covering the substratum on the Biosilon® and Microhex®. Ag8 cells maintained their spherical shapes throughout the culture for all types of microcarriers. In an attempt to scale-up, productions were carried out in 50-ml spinner flasks. Cytodex 3® (for CHO-K1 cells) and Cytoline 2® (for Ag8 cells) were evaluated. The results demonstrate that high yield of biomass could be achieved through the immobilization of the cells in each culture system. And cell cultures on microcarriers, especially on Cytodex 3® and Cytoline 2®, represented a good potential as microcarriers for larger scale cultures of CHO-K1 and Ag8, respectively. Moreover, owing to the fact that the cell lines and culture media are specific, outcomes will be applicable for other clones derived from the same host cell lines.     

小鼠精原干细胞在三种培养基中的生长行为

目的：建立小鼠精原干细胞（SSCs）的体外长期培养体系。方法：用分别添加了等量的胶质细胞源神经营养因子（GDNF）、可溶性GFRα1和hFGF的DMEM／F12、KSR和StemPro-34 SFM三种无血清培养基和MEF饲养层分别培养经差异贴壁分选富集的小鼠SSCs,通过形态观察、标志基因的RT-PCR和免疫细胞化学分析检测其SSCs本原。结果：DMEM／F12与KSR可支持小鼠SSCs在体外存活6-7d,而StemPro-34 SFM能能维持SSCs体外增值一个月。结论：StemPro-34 SFM支持小鼠SSCs的体外增殖。     

CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计

以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分：胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。     

A simple medium for the study of hepatocyte growth in culture under defined conditions

Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation and plating. This work was supported by the Norwegian Cancer Society.     

Adaptation of hybridoma cells to higher ammonia concentration

Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH₄Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH₄Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH₄Cl even under the ammonia serial change.List of symbols D_Feed d^-1 Dilution rate of fresh feed medium (=F_o/V) - D_Out d^-1 Dilution rate of cell suspension (=F₁/V) - F₁ ml·d^-1 Volumetric discharge rate of cell suspension - F₀ ml·d^-1 Volumetric flow rate of fresh feed medium - k_D h^-1 Specific death rate - P mmol·l^-1 Product concentration - S mmol·l^-1 Substrate concentration in culture broth - S₀ mmol·l^-1 Substrate concentration in feed medium - t d Cultivation time - V ml Working volume of reactor - X₀ cells·ml^-1 Total cell density - X_V cells·ml^-1 Viable cell density - Y_P/S mmol·mmol^-1 Yield of product from substrate - Y_X/S cells·mmol^-1 Yield of cells from substrate - mmol·cell^-1·h^-1 Specific production rate - h^-1 Specific growth rate - mmol·cell^-1·h^-1 Specific consumption rate of substrate     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm² by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm². With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

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.     

Water-in-oil microemulsions as the reaction medium for the solvent-sensitive yellow laccases

To determine the applicability of water-in-oil microemulsions for enzymatic conversions catalysed by yellow laccase from Pleurotus ostreatus (YLPO) D1 the following were studied: (i) the catalytic activity of the YLPO D1 in the oxidation of typical phenolic substrates: catechol (CAT), 2,6-dimethoxyphenol (DMOP) and 2,2^'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), at the appropriate pH optimum values in aqueous buffer solutions and in 62 mM bis-2-(ethylhexyl) sulfosuccinate sodium salt (AOT) in isooctane water-in-oil microemulsions; (ii) the effect of acetonitrile (ACN) on the kinetic parameters of DMOP oxidation catalysed by this laccase; (iii) the optimum conditions for the laccase catalytic activity in AOT in isooctane w/o microemulsions (w, laccase, AOT concentration); (iiv) the possibility of using the optimum water-in-oil microemulsions for the oxidation of aromatic alcohols (veratryl alcohol (VA) and benzyl alcohol (BA)) and the oxidative degradation of selected pollutants (3-chlorophenol, anthracene (ANT) and fluorene (FLU)).     

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.