Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco’s modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Intracellular protein degradation in serum-deprived human fibroblasts.

IMR90 human fibroblasts were labelled by incubation of cells for 48 h in medium containing 10% serum and [3H]leucine. The labelled protein was degraded at a rate of 1%/h during a subsequent incubation in medium with 10% serum. Incubation in medium without serum caused a transient enhancement of the degradation of endogenous protein, which was also found in cells labelled in medium without serum. The degradation of micro-injected haemoglobin was enhanced by serum deprivation in a non-transient manner. These results suggest that enhanced degradation in serum-free medium occurs only for a subpopulation of cell proteins and that it appears transient because the major part of the pool of susceptible endogenous proteins is being degraded during the first 20-30 h in serum-free unlabelled medium. Protein turnover in various cell compartments was measured by a double-labelling technique. Most of the enhanced degradation in serum-deprived cultures (73-83%) was due to breakdown of cytosolic proteins. The enhanced degradation of cytosolic proteins seemed to affect several proteins irrespective of their molecular mass or metabolic stability.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.     

Cell-surface modification of non-GMO without chemical treatment by novel GMO-coupled and -separated cocultivation method

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.     

Defined, serum-free culture conditions for the GM-micro-clonogenic assay using agar-capillaries

Culture conditions were determined and optimised for the serum-free growth of granulocyte-macrophage (GM) colonies, derived from mouse bone marrow cells, in agar-containing glass capillaries. The standard medium is DMEM/F-12 (1:1) mixture containing per ml: 10 mg BSA, 35 micrograms transferrin, 20 micrograms soybean lipids and 5 micrograms insulin. In contrast to previous attempts by others, GM-colony yield in serum-free medium was found to be equal to that in serum-containing medium (around 25 colonies/capillary) and to correlate satisfactorily with the cell density at seeding.--The role of polyamine oxidases in cell-proliferation experiments could be studied by this microclonogenic assay without interference from any type of serum.     

Differential survival of cartilage and muscle cells in chick limb-bud cell cultures maintained in chemically defined and serum-containing media

Chick limb buds at stages 22-23 largely consist of replicating presumptive chondroblasts and presumptive myoblasts. To study the influence that different medium compositions may have on the survival, replication, and terminal differentiation of these dissociated cells in vitro, micromass cultures were reared in either standard Dulbecco's modified Eagle's medium containing fetal calf serum (SC-DMEM) or in serum-free DMEM. By day 4, approximately 80% and 50% of the original cell inoculum had been lost in DMEM and SC-DMEM cultures, respectively, as estimated from the recovery of incorporated 3H-thymidine. Between days 1 and 4, the total-DNA content remained virtually constant in DMEM cultures, while it increased five- to sixfold in SC-DMEM cultures. In both media, definitive myoblasts and chondroblasts first emerged on day 1 and day 2, respectively, as determined by immunofluorescence staining using antibodies against muscle light meromyosin (LMM) or the major cartilage proteoglycan. In both media, the chondroblasts increased in number and, by day 4, had formed sizable chondroblast nodules. The number of chondroblasts in SC-DMEM cultures exceeded that observed in DMEM cultures. In DMEM, the LMM-positive myoblasts had an atypical morphology and failed to fuse into elongated myotubes; these cells began to degenerate on about day 4, being undetectable by day 8. In SC-DMEM, the numerous LMM-positive myoblasts located in the center of the micromasses also had an atypical morphology, failed to form multinucleated myotubes, and were absent by day 8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett–Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells.     

A new serum-free medium for monoclonal antibody production

A new serum-free, defined-protein, medium for the growth of murine hybridoma cells and the production of monoclonal antibodies has been developed. Designated WRC 935 medium, this formulation supports the growth of hybridoma cells in higher numbers, and promotes better cell viabilities and increased monoclonal antibody levels compared to growth in DMEM supplemented with 10% fetal bovine serum or in a DMEM/F-12 serum-free mixture. In suspension cultures, WRC 935 medium typically promoted cell growth to densities over two million cells per milliliter. This medium also promoted the rapid growth of cells following their transfer from liquid nitrogen storage. WRC 935 medium is especially useful for high density cell culture production methods using hollow-fiber bioreactors. Hollow-fiber bioreactors using this medium produced antibody at an average rate of 11 mg/day, and the antibody concentration ranged from 10 to 40 mg/ml.     

Beneficial effect of silkworm hemolymph on a CHO cell system: Inhibition of apoptosis and increase of EPO production

To produce erythropoietin (EPO), Chinese hamster ovary (CHO) cells were first cultured in a medium containing FBS (growth medium) and then in a serum-free medium containing sodium butyrate (production medium). Sodium butyrate increases recombinant protein production, but also induces apoptosis, which reduces cell viability and productivity. In a previous study, we found that silkworm hemolymph (SH), an insect serum, inhibits the apoptosis of insect and mammalian cells. To overcome sodium butyrate-induced apoptosis, we added SH to growth medium. This pretreatment with SH inhibited the sodium butyrate-induced apoptosis of CHO cells and consequently increased their longevity and their ability to produce EPO. As a result, the volumetric productivity of EPO was increased five-fold. SH was found to inhibit cytochrome c release from mitochondria into the cytosol, and prevented the activation of caspase-3 and other subsequent caspase reactions.     

CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins

Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.     

Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.     

斜纹夜蛾多角体病毒包埋型病毒受体的鉴定

蔗糖密度梯度离心法提纯斜纹夜蛾核多角体病毒（＄Ｓｐｏｄｏｐｔｅｒａ　ｌｉｔｕｒａ　　ｍｕｌｔｉｎｕｃｌｅｏｃａｐｓｉｄ　ｎｕｃｌｅｏｐｏｌｙｈｄｒｏｖｉｒｕｓ,　ＳｐｌｔＭＮＰＶ）包埋型病毒粒子（ｐｏｌｙｈｅｄｒａ－ｄｅｒｉｖｅｄ　ｖｉｒｕｓ,ＰＤＶ）,以此病毒粒子作抗原,免疫家兔获得抗血清;用ＳＤＳ裂解缓冲液提取斜纹夜蛾幼虫中肠组织细胞总蛋白。采用病毒铺覆蛋白印迹技术（ＶＯＰＢＡ）,利用抗ＰＶＤ抗血清对病毒受体进行检测,结果表明斜纹夜蛾中肠细胞总蛋白中４０ｋＤ、７３ｋＤ、８５ｋＤ的三种蛋白能够结合ＰＤＶ。     

Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and interleukin-6 and granulocyte colony stimulating factor knockout mice

In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50-75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Identification of conditioned medium proteins from human cancer cells adapted to serum-free conditions

Recent studies have shown that human cancer cell lines can be adapted to grow in serum-free, unsupplemented RPMI-1640 (RO) medium. We have developed similar techniques to rapidly identify proteins of interest in serum-free conditioned medium (CM) of human lung cancer cell lines. Classic and variant small cell lung cancer (SCLC) lines were adapted to growth in RO medium. CM from each line was concentrated and fractionated on an anion-exchange column of a fast protein liquid chromatography system. Concentrates of each fraction were loaded onto lanes of minigels of an automated electrophoresis system. Analysis of the chromatograms reveals peaks seen only in CM of the classic SCLC lines. Electrophoretic analysis of the fractions containing these peaks reveal protein bands distinguishing between the subtypes of human SCLC. One protein was purified to homogeneity with subsequent reversed-phase chromatography and identified by protein microsequencing as histone H2B. These automated techniques have general use in the rapid identification of CM proteins associated with the differentiation or progression of the many types of neoplastic cells which can be adapted to growth in RO medium.     

Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Characterization of selenoprotein P as a selenium supply protein.

Selenium (Se) is well known to be essential for cell culture when using a serum-free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se-supplier, T-lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se-containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se-dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P-depleted serum. Compared with other Se-containing proteins, the addition of purified selenoprotein P to the selenoprotein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se-supply protein, delivering Se to the cells.