Anchorage-dependent Cell Line Research Articles

The strategic uses of collagen in adherent cell cultures.

The culture of adherent mammalian cells involves adhesion to the tissue culture vessel. This requires attachment factors from serum and/or a suitable substrate on the vessel surface. Some cells require collagen or other substrates to promote neurite outgrowth, differentiation or growth. However, laboratories often lack guidance on the selection and/or optimisation of collagen. We model such selection/optimisation work in the PC12 neuronal cell line. PC12 (NS-1 variant) cells require a substrate for adherence. Comparing cell attachment against a series of substrates, we found collagen IV to be optimal. We show by comparison of morphology against a range of concentrations that 10 µg/ml is sufficient for supporting cell attachment, and also differentiation. PC12 cells from Riken Cell Bank do not require a substrate for routine culturing but only for differentiation. As all substrates supported attachment equally well, we used a novel serum-free approach and identified collagen IV as its preferred substrate. For these cells, Dulbecco's modified eagle's medium but not Roswell Park Memorial Institute (RPMI)media supports normal cell attachment. However, coating with collagen IV enabled the cells to grow equally well in RPMI. Hence the strategic use of collagen is essential in laboratories working with anchorage-dependent cell lines.

Study of Rotary Cell Culture System-Induced Microgravity Effects on Cancer Biomarkers.

Since humans started to explore the possibilities of life beyond earth, space missions began unmanned in the beginning and late with astronauts to explore the curiosity. There are several factors in space which are different from that of the earth's atmosphere, including gravity, cosmic radiation, extreme temperatures, and heavy ions. The space environment consists of very less gravitational force, which is often termed as microgravity. Microgravity, along with radiation in space, is known to suppress the immune system of astronauts, and hence, the chances of getting diagnosed with cancer could not be overlooked. Microgravity simulators are used to generate microgravity in laboratory conditions, and it could be a very beneficial tool to study the effect of altered gravity on various types of cells, including cancer cells. Cancer cells produce several biomarkers which are used for cancer diagnosis and prognosis. Protein biomarkers are indispensable for the ongoing fight against cancer. In this chapter, we describe the culture of both anchorage-dependent and suspension cell lines using rotary cell culture system (RCCS) to induce microgravity condition and its effect on protein biomarkers.

Nanoparticle technology improves in-vitro attachment of cattle (Bos taurus) trophectoderm cells.

The bovine cell line, cow trophectoderm-1 (CT-1), provides an excellent in-vitro cell culture model to study early embryonic development. Obtaining consistent attachment and outgrowth, however, is difficult because enzymatic disassociation into single cells is detrimental; therefore, CT-1 cells must be passaged in clumps, which do not attach readily to the surface of the dish. We tested whether magnetic nanoparticles, NanoShuttle™-PL, could be used to improve cell attachment and subsequent proliferation of the cattle trophectoderm cell line without altering cellular metabolism or immunofluorescent detection of the lineage marker Caudal Type Homeobox 2 (CDX2). Confluency was achieved more consistently by using the NanoShuttle™-PL system to magnetically force attachment (75-100% of wells) as compared to the control (11%). Moreover, there were no alterations in characteristic morphology, nuclear-localized expression of the trophectoderm marker CDX2, or glycolytic metabolism. By enhancing attachment, magnetic nanoparticles improved culture efficiency and reproducibility in an anchorage-dependent cell line that otherwise was recalcitrant to efficient passaging.

Evaluating the potential of chitosan/poly(vinyl alcohol) membranes as alternative carrier material for proliferation of Vero cells

AbstractChitosan/poly(vinyl alcohol) (CS/PVA) blend membranes were prepared using the casting method and their physiochemical properties were analyzed using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). FTIR and XRD demonstrated possible hydrogen bonds between CS and PVA. The addition of PVA to CS resulted in surface roughness as analyzed by SEM. The CS/PVA blend membrane exhibited high tensile properties (81.62%) and reduced water-holding capacity (53.8%) compared to a pure CS membrane (control). Cell viability and proliferation were assessed via an MTT assay with Vero cell culture. Associated with improved physicochemical properties, the CS/PVA blend membrane promotes cell proliferation of Vero cells with high specific growth rate (0.582 day-1). The results demonstrate that the blending of CS and PVA could significantly alter the surface rugosity, water-holding capacity and improve the mechanical and biological properties of the membrane. Interestingly, this concept can be extended for different anchorage-dependent cell lines, as an alternative carrier material.

Modified Adherence Method (MAM) for Electrofusion of Anchorage-Dependent Cells.

The artificially induced cell fusion is a useful experimental tool in biology, biotechnology and medicine. The electrofusion is a physical method for cell fusion that applies high-voltage electric pulses. The use of electric pulses causes cell membrane structural changes which bring the cell membrane in the so-called fusogenic state. When such fusogenic membranes are in close contact cell fusion takes place. Physical contact between fusion partners can be achieved by various methods and one of them is modified adherence method (MAM) described in detail here on B16-F1 cell line. The method is based on the fact that living cells form contacts in confluent culture. However, instead of using confluent cell culture, in modified adherence method cells are plated in suitable concentration and allowed to form contacts for only short predetermined period of time. During that time the cells are only slightly attached to the dish surface maintaining the spherical shape. Observed high fusion yields up to 50 % obtained by MAM in situ by dual-color fluorescence microscopy are among the highest in field of electrofusion. The method can be readily adapted to other anchorage-dependent cell lines.

Batch, fed‐batch, and microcarrier cultures with CHO cell lines in a pressure‐cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.

Development and strategies of cell-culture technology for influenza vaccine

Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.

Rapid Screening of Serum-Free Media for the Growth of Adherent Vero Cells by Using a Small-Scale and Non-invasive Tool

The paper proposes a rapid screening method for a first step improvement of an animal component-free medium dedicated to the growth of the anchorage-dependent Vero cell line. A new, rapid, and non-invasive technique is presented to specifically monitor cultures of adherent cells in 96-well plates. The operating conditions of an image analyzer are adapted to take into account the decrease of cell size when the attached cell density increases. An experimental design is carried out to assess the influence of ten component groups in the original medium. Two groups including protein extracts, growth factor, insulin, glucose, and pyruvate show significant positive effects. The groups with vitamins and molecules related to nitrogenous bases display a less pronounced influence. The mixture of amino acids, B(1) vitamin, magnesium sulfate, and sodium phosphate as well as the couple sodium citrate and ferric chloride lead to a downward trend. The screening results are proved to be scalable in stirred cultures with cells on microcarriers. An improved serum-free medium, with some component groups being removed or added, can be rapidly formulated to reach respectively similar or 1.6 times higher cell density than in the original medium. The results from this global approach could be helpful to further focus experiments on identified medium components.

Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L6)

In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells, a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L6) was established and the effect of curcumin on biological behavior of Hca-P/L6 was observed. Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential (Hca-P) was subcutaneously inoculated into the medioventral line of a mouse 615 and the first generation of metastatic tumor cells of inguinal lymph node (Hca-P/L1) was obtained. Then, Hca-P/L1 was screened by the route of mouse foot pad subcutaneously → lymph node → scale-up culture in vitro → mouse foot pad subcutaneously for five times consecutively. The sensitivity of two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L6) and two anchorage-dependent human hepatocarcinoma cell lines (SMC7721 and HepG2) to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after these cells had been pretreated by curcumin at the concentration of 15–240 μmol/L for 48 h. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 μmol/L in vitro, the effect of curcumin against cell proliferation of Hca-P and Hca-P/L6 was observed by inverted microscope, cell growth curve and cell population doubling time; the effects of curcumin on cell cycles of Hca-P/L6 and Hca-P were studied by flow cytometry (FCM). The results showed Hca-P/L6 spreading to the lymph nodes at multiple sites in mice was screened from Hca-P. The lymph node metastatic rate was 100%. Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner (P<0. 05). At concentrations of 30–120 μmol/L, curcumin had more inhibition on murine ascites hepatocarcinoma cell lines than on human anchorage-dependent hepatocarcinoma cell lines. At concentrations of 60–240 μmol/L, curcumin had more inhibition on Hca-P/L6 with the 50% inhibitory concentration (IC50) of 51.48 μmol/L than on Hca-P with IC50 of 90.87 μmol/L. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days, the proliferations of Hca-P/L6 and Hca-P were inhibited (P<0.05) in a time-dependent manner (P<0.01) and the population doubling time of Hca-P/L6 and Hca-P was prolonged (P<0.01), and curcumin had more inhibition on Hca-P/L6 than on Hca-P (P<0.05). After pretreatment by 15 μmol/L curcumin for 48 h, the morphous of Hca-P/L6 was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L6 being blocked in the S phase and Hca-P in the S and G2/M phases. Hca-P/L6 was validated to be more sensitive to curcumin than Hca-P. Hca-P/L6 is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.

Human cell culture process capability: a comparison of manual and automated production

Cell culture is one of the critical bioprocessing steps required to generate sufficient human-derived cellular material for most cell-based therapeutic applications in regenerative medicine. Automated cell expansion is fundamental to the development of scaled, robust and cost effective commercial production processes for cell-based therapeutic products. This paper describes the first application of process capability analysis to establish and compare the short-term process capability of manual and automated processes for the in vitro expansion of a selected anchorage-dependent cell line. Estimates of the process capability indices (Cp, Cpk) have been used to assess the ability of both processes to consistently meet the requirements for a selected productivity output and to direct process improvement activities. Point estimates of Cp and Cpk show that the manual process has poor capability (Cp = 0.55, Cpk = 0.26) compared to the automated process (Cp = 1.32, Cpk = 0.25), resulting from excess variability. Comparison of point estimates, which shows that Cpk < Cp, indicates that the automated process mean was off-centre and that intervention is required to adjust the location of the process mean. A process improvement strategy involving an adjustment to the automated process settings has demonstrated in principle that the process mean can be shifted closer to the centre of the specification to achieve an estimated seven-fold improvement in process performance. In practice, the 90% confidence bound estimate of Cp (Cp = 0.90) indicates that that once the process is centred within the specification, a further reduction of process variation is required to attain an automated process with the desired minimum capability requirement.

A GFP-transfected HFWT cell line, GHINK-1, as a novel target for non-RI activated natural killer cytotoxicity assay.

An anchorage-dependent Wilms' tumor cell line, HFWT, has been found to be extremely susceptible to human natural killer (NK) cells. Here we established a transfectant of HFWT with the green fluorescence protein (GFP) gene, designated GHINK-1 cells, to apply to the activated NK cytotoxicity assay without radioisotope labeling. After being co-cultured with CD3 CD56+ NK cells, GHINK-1 cells released GFP into the medium. The intensity of the fluorescence from the released GFP correlated almost exactly with the radioactivity of a standard 51Cr-release assay performed with suspension-cultured K562 cells. Because it does not require separation of the remaining live target cells by centrifugation, the non-radioisotopic GFP release assay with GHINK-1 cells is a convenient alternative for monitoring human activated NK killing activity.

The role of Bcl-2 and its combined effect with p21CIP1 in adaptation of CHO cells to suspension and protein-free culture

The overexpression of the antiapoptotic gene Bcl-2 has been previously shown to protect cells from undergoing apoptosis during exposure to environmental stress. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. To determine if the overexpression of Bcl-2 could improve the process of adaptation to suspension and protein-free growth environments, we have studied the growth and viability of anchorage-dependent Chinese hamster ovary cell lines that differ only in there expression of Bcl-2. In addition, we examined the effect of combining Bcl-2 and p21CIP1 expression during adaptation to suspension and protein-free environments. The results of this study provide evidence of a clear reduction in the overall time required for the process of adaptation to both suspension and protein-free environments in Bcl-2 expressing cultures and that through the combined expression of p21CIP1 and Bcl-2, it is possible to further reduce the time. The Bcl-2 results support the well-demonstrated concept that this protein plays an important role in apoptotic signaling pathways and suggest that it may also provide more diverse functions beside its death-inhibiting role.

Surface modified poly(L‐lactide‐co‐ε‐caprolactone) microspheres as scaffold for tissue engineering

P-15 modified poly(L-lactide-co-epsilon-caprolactone) (PLCL) microspheres were investigated as scaffolds for tissue engineering applications. PLCL copolymer was synthesized by ring opening polymerization and was composed of a soft matrix of mainly epsilon-caprolactone moieties and hard domains containing more of L-lactide units thus exhibiting a rubber like elasticity responsible for providing mechanical strength to scaffolds. Microspheres were fabricated by solvent evaporation method and surface modified with P-15, a synthetic analogue of collagen. These were then evaluated for cell adhesion, ECM formation and cell proliferation. Anchorage dependent cell lines LLCPK-1 and L6 were seeded on PLCL microspheres (unmodified surface activated microspheres) and P-15-PLCL microspheres (P-15 modified microspheres). P-15 modified microspheres showed significantly higher cell adhesion and viability than unmodified microspheres. Scanning electron microscopy also revealed copious amount of extra-cellular matrix production by P-15. Initial results of cell culture experiment on two different cell lines suggested that the growth of LLCPK-1 in a 3D environment with P-15 modified microspheres is via spreading and flattening on the surface of scaffold followed by formation of a sheet-like structure while L6 grew in the form of multilayered structures with formation of interparticulate cellular bridges. The 3D moldable nature, combined with modification of surface chemistry with cell adhesion molecules such as P-15 to enhance proliferation of epithelial cells and myoblasts, recommends further investigation of P-15-PLCL microspheres for production of an ideal scaffold for soft tissue engineering.

Bioreactor systems for the production of biopharmaceuticals from animal cells

The demand for biopharmaceutical products is set to see a significant increase over the next few years. As a consequence, the processes used to produce these products must be able to meet market requirements. The present paper reviews the current technologies available for animal cell culture and highlights the advantages and disadvantages of each method, while also providing details of recent case studies. Processes are described for both suspension and anchorage-dependent cell lines.

Transformation by inorganic arsenic compounds of normal Syrian hamster embryo cells into a neoplastic state in which they become anchorage-independent and cause tumors in newborn hamsters.

Arsenic is a known human carcinogen, but little evidence exists for its carcinogenicity in animals. In order to investigate the ability of inorganic arsenics to transform normal cells into a neoplastic state, mass cultures of normal, diploid Syrian hamster embryo (SHE) cells exposed to various concentrations of sodium arsenite or sodium arsenate for 48 hr were continually passaged and tested for neoplastic transformation, as determined by anchorage-independent growth in semisolid agar and tumorigenicity in newborn hamsters. Twenty-one of 22 (96%) untreated, control cultures senesced by 20 passages. While 1 culture escaped senescence, it did not acquire the ability to either grow in semisolid agar or form tumors in animals. Ten of 14 (71%) cultures exposed to sodium arsenite or sodium arsenate escaped senescence. Nine of the 10 (90%) arsenic-treated immortal cultures acquired the anchorage-independent phenotype. Five of 5 anchorage-independent cultures examined were tumorigenic. Two of 3 morphologically transformed colonies induced by sodium arsenate also acquired the ability to grow in semisolid agar when isolated. Amplification of the c-myc or c-Ha-ras oncogene was detected in 3 of 5 and 4 of 5 tumorigenic cell lines, respectively. Both c-myc and c-Ha-ras were amplified even in a preneoplastic, anchorage-dependent cell line, but neither was amplified in 6 of 9 anchorage-independent cell lines. Overexpression of c-myc and c-Ha-ras mRNA was observed in most of the neoplastically transformed cell lines but not in the preneoplastic cell line. Experiments using the methylation-sensitive restriction endonuclease isoschizomers HpaII and MspI revealed hypomethylation of c-myc and c-Ha-ras in the 5'-CCGG sequence of arsenic-exposed cell lines but not in the parental SHE cells or a spontaneously transformed cell line. Thus, inorganic arsenics induce neoplastic transformation of normal, diploid mammalian cells. Overexpression of oncogenes by DNA hypomethylation may participate in the arsenic-induced neoplastic transformation of mammalian cells.

Impact of cell growth morphology on retroviral transduction: effect of contact inhibition.

Retrovirus-mediated gene transfer is one of the most commonly used methods to deliver, integrate, and express the gene of interest because the retrovirus can insert the desired gene into the chromosome of the target cells with high stability. However, to deliver the gene successfully, the retrovirus requires active division to integrate reversely transcribed DNA into the chromosome of target cells. In this study, we focused on the effect of cell-cell contact inhibition on the efficiency of retroviral transduction with two anchorage-dependent cell lines: NIH 3T3 and 293 cells. These two cell lines have very different cell morphologies and growth patterns on surfaces. Human embryonic kidney epithelial 293 cells tend to stick together after dividing, while NIH 3T3 cells migrate to occupy available surface and spread. Experimental data indicate that the abatement of the transduction rate of 293 cells was initiated in the early stage of the culture, whereas effect of contact inhibition of NIH 3T3 cells on the transduction rate became dominating at the end of the culture period. Experimental results were also quantitatively illustrated by plotting normalized multiplicity of infection (MOI) versus normalized cell density. According to the outcomes, cell inoculation density plays an important role in optimizing the retroviral transduction rate. The optimal time of retroviral transduction should be confined to the accelerating growth phase for 293 cells and at the exponential growth phase for NIH 3T3 cells. The implication drawn from this study is that contact inhibition effect on retroviral transduction should be taken into account for large-scale gene transfer systems such as the microcarrier bioreactor.

Effect of insulin stimulation on the proliferation and death of Chinese hamster ovary cells.

The effect of environmental and genetic parameters on cell death was studied in Chinese hamster ovary cell cultures. Experiments were performed using an anchorage-dependent CHO cell line expressing gamma-IFN, and a second cell line obtained by transfection of the previous one with bcl-2. In serum-free medium the two cell lines showed a considerable degree of growth control entering quiescence while maintaining high viabilities. The addition of transferrin did not have any effect but insulin addition allowed cells arrested by serum withdrawal to reenter the cell cycle. However, insulin supplementation also resulted in cell death, which was possible to avoid through bcl-2 overexpression or in the presence of serum. We propose that the expression of c-myc, shown to be induced by insulin, plays an important role in the cell death observed after insulin addition in an inappropriate environment, deficient in protective factors. This hypothesis is supported by measurements of c-myc expression and cell cycle distribution.

Production of urokinase by HT 1080 human kidney cell line.

Studies were carried out in T-flasks and bioreactor to produce urokinase enzyme using HT 1080 human kidney cell line. While growing the cell line it has been observed that the lag phase is reduced considerably in the bioreactor as compared to T-flask culture. The HT 1080 cell adhesion rate and urokinase production were observed to be the function of serum concentration in the medium. The maximum urokinase activity of 3.1 x 10(-4) unit ml(-1) was achieved in the bioreactor at around 65 h of batch culture. Since HT 1080 is an anchorage dependent cell line, therefore, the hydrodynamic effects on the cell line were investigated.

A microcarrier-based cell culture process for the production of a bovine respiratory syncytial virus vaccine.

Veterinary viral vaccines generally comprise either attenuated or chemically inactivated viruses which have been propagated on mammalian cell substrates or specific pathogen free (SPF) eggs. New generation vaccines include chemically inactivated virally-infected whole cell vaccines. The NM57 cell line is a bovine nasal turbinate persistently infected (non-lytic infection) with a strain of the respiratory syncytial virus (RSV). The potential of microcarrier technology for the cultivation in bioreactors of this anchorage dependent cell line for RSV vaccine production has been investigated. Both Cytodex 3 and Cultispher S microcarriers proved most suitable from a selection of microcarriers as growth substrates for this NM57 cell line. Maximum cell densities of 4.12×105 cells ml-1and 5.52×105 cells ml-1 respectively were obtained using Cytodex 3 (3 g l-1) and and Cultispher S (1 g l-1) in 5 l bioreactor cultures. The fact that cell growth was less sensitive to agitation rate when cultured on Cultispher S microcarriers, and that cells were efficiently harvested from this microcarrier by an enzymatic method, suggested Cultispher S is suitable for further evaluation at larger bioreactor scales (>5 l) than that described here.

The human complement regulatory factor-H-like protein 1, which represents a truncated form of factor H, displays cell-attachment activity.

Complement factor H (FH) and factor-H-like protein 1 (FHL-1) are human plasma proteins with regulatory functions in the alternative pathway of complement activation. FH and FHL-1 are organized in repetitive elements termed short consensus repeats (SCRs) and the seven SCRs of FHL-1 are identical with the N-terminal domain of the 20 SCRs of FH. The fourth SCR of both proteins (SCR 4) includes the sequence Arg-Gly-Asp (RGD), a motif that is responsible for the major adhesive activity of matrix proteins like fibronectin. A synthetic hexapeptide with the sequence ERGDAV derived from the RGD domain of FH/FHL-1 interferes with cell attachment to a fibronectin matrix. Although the identical motif is present in both FH and FHL-1, only FHL-1 acts as a matrix for cell spreading and attachment, thus the two proteins differ in function. The adhesive activity of FHL-1 is localized to the RGD-containing SCR 4 by the use of recombinant fragments. All three analysed anchorage-dependent cell lines (CCl64, C32 and MRC-5) adhere to an FHL-1 matrix. The use of synthetic peptides in competition assays, on either FHL-1-derived or fibronectin matrices, shows that the cellular receptors binding to the FH/FHL-1-derived RGD motif are related to or identical with integrin receptors which interact with fibronectin. The identification of a functional adhesive domain in the FH/FHL-1 sequence demonstrates, at least for FHL-1, a role in cell attachment and adhesion.