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

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

Enhanced retroviral transduction of 293 cells cultured on liquid-liquid interfaces

In clinical research, retrovirus-mediated gene therapy is one of the most commonly used methods to deliver and express the gene of interest due its ability to allow for stable gene integration into the chromosomes of target cells. To elevate the efficiency of viral transduction, several restrictions, such as low virus-cell encounters and the necessity for cell division, must be improved. In this study, we focused on the possibility of accelerating cell division and the ensuing increment of viral transduction on flexible substrata. Perfluorocarbon FC-40 was harnessed to form a liquid-liquid interface with culture medium. Enhanced green fluorescence protein (EGFP) was employed as the marker gene to quickly illustrate the percentage of viral infection. The results indicate that the gene transfer efficiency to 293 cells cultured on protein-precoated liquid-liquid interfaces was higher than in cells cultured on rigid polystyrene surfaces. This increased transduction rate on the liquid-liquid interface is consistent with the acceleration of division of 293 cells on a flexible interface, which exhibited less adhesiveness. The effect of cell-cell contact inhibition on the rate of gene transduction is also addressed in this study.     

Anti-steroid action in cultured L-929 mouse fibroblasts

L-929 mouse fibroblast growth is arrested by the glucocorticosteroid dexamethasone (dex), which also decreases adhesiveness to culture plates. Both dex effects were abolished when RU 486, a new synthetic anti-hormonal steroid, was added to the culture medium. Using [3H]RU 486, binding studies revealed that RU 486 bound approximately 25,000 sites/cell of the glucocorticosteroid receptor (GR) with affinity higher than that of dex and translocated GR to the nucleus. However, the distribution of steroid-receptor complexes between cytosol and nuclei was different for the agonist and the antagonist, with more nuclear accumulation in the case of dex. Estradiol increases L-929 cell growth and adhesiveness to culture plates, but not if the anti-estrogen tamoxifen (tam) was added. These observations initially made in serum containing medium, were confirmed in serum-free, chemically defined culture medium (SF). In SF medium, tam (1 microM) provoked death of most L-929 cells after 10 days of culture, leading to the selection of a variant clone LB of tam-resistant cells. Tam binds to the estrogen receptor (ER), but with less affinity than estradiol. ER concentration, estimated by the binding of 4-hydroxytamoxifen (OH-tam) and of estradiol was lower in LB cells than in original tam-sensitive L-929 cells. The concentration of specific anti-estrogen binding sites in the particulate fraction of the cells, measured by OH-tam binding, non competed by estradiol, was also significantly diminished in tam-resistant LB cells.     

Affinity capture of proteins from solution and their dissociation by contact printing

Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (alphaCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.     

Adhesion of cells to polystyrene surfaces

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.     

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.     

Hepatocyte culture on biodegradable polymeric substrates

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 mug/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.     

Autoclavable low cost serum-free cell culture media. The growth of L Cells and BHK cells on peptones

The growth of L-929 cells on a series of peptones, and protein hydrolysates was examined, and it was found that when MEM was supplemented with any of a series of peptones, cell growth was about as good as when serum was used as a supplement. Protein hydrolysates did not support cell growth very well and at higher concentrations actually reduced cell growth. L-929 and L-60TM cells were grown both as monolayers or stationary suspensions and in agitated systems in MEM supplemented with 0.5—1% bactopeptone. The addition of macromolecular compounds, insulin or oleic acid had no effect on cell growth. BHK cells were also grown on media supplemented with bactopeptone but richer media (MEM-alpha, F-12, or RMPI1640) gave higher cell yields. The cells did not form the monolayers observed with fetal calf serum, but a partial suspension system. Addition of a detergent Darvan #2 gave a totally suspension culture in both stationary and agitated systems. The production of Sindbis virus in BHK cells grown in serum-free media was examined and the yield of virus was found to be about the same as that produced in serum-supplemented systems. It is estimated that the cost of cell production media could be reduced by about 90% by the replacement of serum supplement by peptones.     

The kinetics of Lagenidium giganteum growth in liquid and solid cultures

AIMS: Production of the mosquito biolarvacide Lagenidium giganteum in solid culture has been proposed as an economic alternative to production in liquid culture because of observations of improved shelf life and efficacy upon storage. Understanding the differences between these production systems and estimating growth rate in solid culture are important for commercialization. In order to address these needs a logistic model was developed to describe the growth kinetics of L. giganteum produced in solid and liquid cultures. METHODS AND RESULTS: Kinetic parameters in the logistic model were estimated by nonlinear regression of CO2 evolution rate (CER) and biomass data from solid and liquid cultivation experiments. Lagenidium giganteum biomass was measured using DNA extracted directly from samples. The logistic model was fit to experimental biomass and CER data with low standard errors for parameter estimates. The model was validated in two independent experiments by examining prediction of biomass using on-line CER measurements. CONCLUSIONS: There were significant differences between maximum biomass density, maintenance coefficients, and specific growth rates for liquid and solid cultures. The maximum biomass density (mg dw ml-1) was 11 times greater for solid cultivation compared with liquid cultivation of L. giganteum; however, the maintenance coefficient (mg CO2 h-1 (mg dw)-1) was six times greater for liquid cultivation than in solid cultivation. The specific growth rate at 30 degrees C was approximately 30% greater in liquid cultivation compared with solid cultivation. Slower depletion of substrate and lower endogenous metabolism may explain the longer shelf life of L. giganteum produced in solid culture. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple logistic model was developed which allows real-time estimation of L. giganteum biomass from on-line CER measurements. Parameter estimates for liquid and solid cultivation models also elucidated observations of longer shelf life for production in solid culture.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium.

The growth of L-60TM cells (a suspension culture adapted L-cell) on media composed of MEM (minimum essential medium (Eagle)) and bactopeptone autoclaved together or separately under a variety of conditions has veen determined. It has been found that MEM autoclaved with 0.5% bactopeptone at 15 psi for 20 min, cooled and then neutralized with NaHCO3, consistently supported good cell growth of L-60TM and L-929 cells. Similar results were obtained when the MEM and bactopeptone were autoclaved separately. The cells grew initially as a monolayer, subsequently becoming a stationary suspension. Some experiments were carried out with agitated suspension culture of L-60TM cells in the autoclaved MEM-bactopeptone combination with and without added methylcellulose and results were obtained which indicate that large scale suspension culture is possible in this system. Other peptones were also found to support cell growth. The autoclaved MEM-bactopeptone combination also supported the growth of Chang liver and Vero cells. The Chang liver cells rapidly dissociated from the plastic surface but the Vero cells remained sufficiently securely attached so that it was possible to grow them near to confluency in roller bottles.     

Use of a gelatin substrate for culturing diploid human skin fibroblasts

Gelatin coating of the growth surface of commercially available polystyrene Petri dishes is proposed for successful cultivation of human skin fibroblasts. A comparison of culture growth dynamics on gelatin, glass and polystyrene allowed to recommend gelatin as the substrate in order to receive an abundant similar material with a low population density viability. The absence of changes in carbohydrate metabolism changes during cell culturing on gelatin provides an opportunity to use these cells in studying regulation of carbohydrate metabolism.     

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.     

Role of divalent cations, pH, cytoskeleton components and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate

The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed. The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended. The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6. The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion. Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion. Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.     

Effects of sex steroids and antihormones on growth, adhesiveness and receptors of L-929 cells cultured in serum-containing and serum-free media.

L-929 cells contain distinct steroid hormone receptors for glucocorticosteroids, for androgens and for estrogens. We studied the effects of different hormones at physiological concentrations on androgen and estrogen receptor protein accumulation and on cell multiplication. The cells were cultured in steroid-free serum-containing medium, either in Petri dishes or in suspension cultures, and in serum-free medium in Petri dishes. The presence of androstanolone (30 nM) in suspension cultures decreased the concentration of estradiol receptor-binding sites in the cytoplasmic fraction. This decrease was progressive following 3, 5 or 10 days of suspension culture in the presence of the androgen; simultaneously a parallel increase in cell multiplication and DNA was observed. The estradiol receptor decrease was approx. 50% after 10 days of treatment and was unaltered after a further 5 days. It was verified that the low androstanolone concentration in the medium did not provoke the translocation of the estradiol receptor into the nucleus. Progesterone 50 nM also decreased the cytoplasmic estradiol binding sites but had no influence on cell growth and no cytoplasmic progesterone receptor could be found. Diethylstilbestrol (30 nM) did not decrease the concentration of androgen receptor.Cell multiplication was stimulated after several days of suspension culture in the presence of either diethylstilbestrol, estradiol or androstanolone at a concentration of 10–30 nM. The specific anti-hormones, tamoxifen and cyproterone acetate, inhibited selectively the growth effects of estrogens and androgen, respectively. L-929 cells could be cultured for a long period of time in serum-free medium in Petri dishes. Cell adhesiveness was increased in the presence of 40 nM androstanolone or 40 nM estradiol, as well as cell multiplication. Dexamethasone had a negative effect on cell adhesiveness and cell growth. The experimental data suggest that at low concentrations the different steroids operated each through its own receptor and were active on cell growth even in serum-free medium.     

Thermodynamic aspects of plant cell adhesion to polymer surfaces

Summary A thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm² which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm²) agree with the pattern of adhesion previously described.     

Extracellular protease as a reversible adhesion regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2-3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h-1, the fraction of proteins in cells being almost the same (60-70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3-5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

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

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

Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA

Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1-, S-, and G2-Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7-48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L-929 cells in medium without serum reduced their RNA content down to one-third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.     

Mouse Fibroblast Cell Adhesion Studied by Neutron Reflectometry

Neutron reflectometry (NR) was used to examine live mouse fibroblast cells adherent on a quartz substrate in a deuterated phosphate-buffered saline environment at room temperature. These measurements represent the first, to our knowledge, successful visualization and quantization of the interface between live cells and a substrate with subnanometer resolution using NR. NR data, attributable to the adhesion of live cells, were observed and compared with data from pure growth medium. Independently of surface cell density, the average distance between the center of the cell membrane region and the quartz substrate was determined to be ∼180 Å. The membrane region (∼80 Å thick) contains the membranes of cells that are inhomogeneously distributed or undulating, likely conforming to the nonplanar geometry of the supporting adherence proteins. A second region of cell membranes at a greater distance from the substrate was not detectable by NR due to the resolution limits of the technique employed. Attachment of the live cell samples was confirmed by interaction with both distilled water and trypsin. Distinct changes in the NR data after exposure indicate the removal of cells from the substrate.