Suspension culture of drosophila cells employing a gyratory shaker

Summary To develop a method for culturing a large number of small-scale suspension cultures ofDrosophila melanogaster cells simultaneously, basic conditions were studied using a cell line GM₂ and a gyratory shaker. Under gyration at more than 180 rpm, a majority (>80%) of the cells still remained as suspension and grew normally. Lower speed of gyration caused adhesion of the cells to a substratum. Furthermore, size of the culture vessels was found to affect the pattern of cell growth. Five- or 10-ml Erlenmeyer flasks gave satisfactory results, but the growth curves in 30-ml flasks differed from flask to flask and the saturation level was lower. Besides, the growth curves in the latter case were quite different depending on the volume of the medium. A preliminary experiment showed that the type of flask might affect the pattern of a growth curve. Initial cell densities has to be more than 6×10⁴ cells per ml. Lower densities resulted in the longer doubling time or no increase in the cell number. Therefore the following conditions are recommended as a standard for gyration culture ofD. melanogaster cell, GM₂: speed of gyration, 180 rpm; culture vessel, 5- or 10-ml Erlenmeyer flask of a certain type; initial cell density, 1 to 5×10⁵ per ml. Both D20 and modified Schneider’s medium could be utilized as the medium.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Improving the methods for isolation of monocyte and establishing macrophage cell culture in caprine model

Monocytes are widely used for immunological research, especially in the study of innate immune system. Although methods for isolation of human monocytes have been established, the procedure for non-human monocyte has not been well developed. This paper describes an improved method for isolation of monocyte and the subsequent macrophage cultivation from caprine blood. Monocytes were isolated from 16 ml of heparinized caprine blood using double density methods; the Ficoll and Percoll. The number of monocytes obtained was 5.12 ± 0.89 × 10⁷ cells/ml at 70 % purity. The isolated monocytes were maintained in 10 % fetal bovine serum-enriched Dulbecco’s Modified Eagle Medium for maturation to form macrophage cell culture. At the end of the experiment, the harvested macrophage was 2.48 ± 0.33 × 10⁶ cells/ml.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Optimization of a MRC-5 Cell Culture Process for the Production of a Smallpox Vaccine

A cell culture process adaptable to produce smallpox vaccine at large scale has been developed. To achieve this, Design of Experiments (DOE) was applied to identify and optimize critical cell culture process parameters for MRC-5 cell growth and recovery during cell expansion. For cell growth, a 2^5?1 partial factorial (two level, five factor, 16 conditions) study was designed to evaluate the effects of basal media, seeding density, culture volume, feeding frequency and serum concentration on population doubling level (PDL) after 6–7 days in adherent T-flask cultures. Results indicated that lowering the cell density to 1×10⁴ cells/cm², increasing the culture volume to 0.5 ml/cm² and increasing serum concentration to 20% significantly improved cell expansion. These findings correlated with PDLs above 2.0 and cell densities above 1×10⁵ cells/cm² at the end of the study period. For cell recovery at passaging, a similar DOE was used to evaluate the effect of trypsin concentration, solution temperature, duration of treatment, incubation temperature and duration of standing time between quenching and reseeding. By increasing the trypsin treatment duration to 60 min and lowering the standing time between quenching and reseeding to within 1 h, the recovery of the MRC-5 cells was greatly improved. By using these newly defined conditions, a two-fold improvement in cell expansion was consistently achieved in both roller bottles and 10 layer Nunc^® Cell Factories (Cell Factories). Application of these new conditions for current Good Manufacturing Practices (cGMP) production of MRC-5 cell banks and clinical material demonstrated predictably high cell expansion as well as significantly higher production of vaccinia virus, thus providing the basis for manufacturing vaccinia virus at large scale. These findings demonstrate the need for cell culture optimization and the effectiveness of DOE to rapidly define processes suitable for cGMP manufacturing of a smallpox vaccine or other viral vaccine products.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> cell elicitor enhances betalain content in the cell suspension culture of <Emphasis Type="Italic">Celosia cristata</Emphasis>

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30 ml MS media fortified with 13.5 μM 2,4-D and 0.44 μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125 × 10^?3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.     

Large-scale production of Erythroid Differentiation Factor (EDF) by gene-engineered Chinese hamster ovary (CHO) cells in suspension culture

From Chinese hamster ovary (CHO) cells which were producing Erythroid Differentiation Factor (EDF) in a culture medium, anchorage-independent cells, named as CHO-SPN, were produced by repeated cultivating in a suspension system. The growth time and maximum cell density of the CHO-SPN cells were 48 h and 7.8×10⁵ viable cells/ml. CHO-SPN cells accumulated 8,000 units/ml (corresponding to 4 mg/ml) of EDF in 4d. After 20 cycles of culture, CHO-SPN cells still possessed the same EDF productivity and the same growth kinetics. Furthermore, in an appropriate dissolved oxygen concentration and pH controlled culture system, the growth time and cell density became 24 h and 1×10⁶ viable cells/ml. The critical level of dissolved oxygen for cell growth was 0.015 atm. The maximum oxygen demand was 3.3×10⁻⁹ mole of O₂/ml/min.Fetal bovine serum (FBS) was indispensable for cell growth. However, a FBS-free medium (ASF201) was available for maintenance of the CHO-SPN cells, and EDF production occurred in the same medium.     

Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells

In laboratory scale therapeutical protein production, cell clumps form typically in shake flasks, which hinders cell growth and decreases protein yield. To minimize clumps during the culture of Chinese hamster ovary cells, we employed the combination of two reagents, dextran sulfate (DS) and recombinant trypsin (r-trypsin). Our results showed that both DS and r-trypsin could diminish cell aggregation when adding them respectively, but clumps were still noticed obviously. In order to further mitigate cell agglomerate, a combination of 1.2 g/L DS and 8.0 mg/L r-trypsin was employed and no clumps were found under the bright field microscope. Strikingly, the highest viable cell density of combination group was increased from 5.12 × 10⁶ to 7.13 × 10⁶ cells/mL, while the integral of viable cells concentration was raised from 35.13 × 10⁶ to 60.87 × 10⁶ cells·days/mL, and the culture period was prolonged by 4 days. In addition, the antibody integrity was maintained in the combination group compared with that of the control.     

Expansion of mouse sertoli cells on microcarriers

Background: Sertoli cells (SCs) have been described as the ‘nurse cells’ of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up. Objective: Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large‐scale culture. Materials and methods: In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture. Results: After 6 days, maximal cell densities achieved were 4.6 × 10⁶ cells/ml for Cytodex‐1 in DMEM/FBS compared to 4.8 × 10⁵ cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 × 10⁵ cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. Results indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co‐culturing with NSCs and immunostaining. Conclusion: Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost‐effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time‐consuming and susceptible to contamination.     

Establishment of human interleukin-4 producing Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were transfected with a human interleukin 4 (IL-4) expression plasmid in which human IL-4 cDNA is linked downstream of the human cytomegalovirus/human immunodeficiency virus chimeric promoter. The plasmid also contained a mouse dihydrofolate reductase (dhfr) gene, expression of which is directed by the SV40 early promoter. The resulting methotrexate-resistant, transformed cells constitutively secreted a high level of human IL-4. CHO cells producing human IL-4 were cultured on microcarriers in a perfusion cell culture system containing 1 l of culture medium, and a high level of human IL-4 (5 × 10⁴U ml⁻¹) was produced at a high cell density (1 × 10⁷cells per ml). Serum-free culture was also examined.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Potent inhibition of cell density-dependent apoptosis and enhancement of survival by dimethyl sulfoxide in human myeloblastic HL-60 cells

Human myeloblastic cell line HL-60 cells undergo apoptosis during in vitro culture in a cell density-dependent manner, and this cell density-dependent apoptosis was observed when the concentration of cultured cells exceeded 8–10 × 10⁵ cells/ml. Dimethyl sulfoxide (DMSO), a differentiation inducer of HL-60 cells, did not amplify, but rather potently inhibited, this apoptosis. In a low density culture condition, DMSO attenuated proliferation of HL-60 cells in spite of its inhibition of apoptosis. In contrast, DMSO did support cell survival under high cell density conditions, and DMSO-treated HL-60 cells reached an extremely high concentration of 2–3 × 10⁶ cells/ml, a condition which could never be possible in a usual culture environment. Thus, DMSO exerted dual effects on cell proliferation, i.e., growth inhibition and apoptosis inhibition, and the sum of these effects resulted in an apparently distinct phenomenon according to the culture conditions including cell density. J. Cell. Physiol. 174:135–143, 1998. © 1998 Wiley-Liss, Inc.     

Starvation survival of Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT7137, exopolysaccharide-producing bacterium, was isolated from groundwater contaminated with leachate in a former municipal landfill site (São Paulo, Brazil). The strain was submitted to starvation in phosphate-buffered saline solution for 56 days so as to evaluate its behavior regarding cuturability and cell morphology. As a response to starvation, G. polyisoprenivorans CCT7137 presented reduction in viable cell count, cell size and cell shape alteration. The initial number of viable cells was 1.51 × 10⁷ c.f.u. ml. After 7 days of starvation culturability dropped to 13.70% (2.07 × 10⁶ c.f.u/ml) and, after 56 days, to 3.25% (4.93 × 10⁵ c.f.u/ml). It was also observed that after 7 days of starvation the cell size presented an average reduction in the values of length, length/width ratio, volume and area of 50, 58, 40 and 42%, respectively. The length/width ratio showed a change of shape from rod to coccobacillus. Changes in cell dimensions and distribution of cell into classes were not significant after day 7 of starvation. The results obtained show that it is not necessary for the strain to starve for more than a week to obtain G. polyisoprenivorans CCT7137 size- reduced cells. The results also indicate the potential for its starved forms to be used in future tests in porous medium to study the production of exopolysaccharide in situ.     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.     

Propagation of Autographa californica nuclear polyhedrosis virus in cell culture: Methods for infecting cells

Autographa californica nuclear polyhedrosis virus (AcNPV) produced in Trichoplusia ni (TN-368) cells was used to infect other cell cultures. Methods were developed to recover and obtain high titers of virus from infected cells for subsequent use as inocula. To release cell-associated nucleocapsids, the cells were lysed by sonication and freeze-thawing. The infectivity of enveloped nucleocapsids was greatly reduced by freeze-thawing, while sonication was not as detrimental. The titer of plaque-forming units (pfu) was reduced about 12-fold when passed through 0.45-μm filters. The virus and cells were manipulated to determine the most efficient methods for inoculating cells while yielding the highest numbers of polyhedra. The viral inocula may be left on cells during virus replication, and cells may be centrifuged at 380 g prior to exposure to virus without affecting the yield of polyhedra. The production of polyhedra is affected by cell density, and, of the densities tested, 7.65 × 10⁵ cells/ml yielded the maximum number of polyhedra per cell (142). However, the highest number of polyhedra per milliliter of culture (2.2 × 10⁸) was obtained with 3.8 × 10⁶ cells/ml. The numbers of polyhedra per cell did not vary when cells were taken from fermentor cultures at 0–144 hr and were infected with virus.     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Continuous insect cell (Sf-9) culture with aeration through sparging

The continuous growth of Spodoptera frugiperda Sf-9 cells in a 250-ml blown-glass jacketed spinner flask under a direct air sparging environment was investigated. Even at 220 ml working volume (about 90% of total volume), this spinner flask provided good mixing and oxygenation as demonstrated by a higher cell density compared with fermentor cultures. This eliminates a common limitation of the traditional spinner flask, namely much lower cell density at high working volume. Furthermore, this spinner flask has been run with Sf-9 cell culture at five different dilution rates and two different air sparging rates at steady state, demonstrating its utility in research applications where cell size, metabolic activity and environmental conditions can be constantly maintained. In addition to demonstrating the utility of the reactor, three novel points are made in this report. First, cell density in continuous cultures is increased significantly due to a high agitation rate and, especially, air sparging rate, which is seldom used in animal cell or insect cell culture. Second, there is no apparent difference in the specific death rate at two different sparging rates (0.0093 vvm and 0.0125 vvm). Finally, we have maintained Sf-9 cells for more than 4 months in a continuous culture using a serum-free medium without loss of recombinant protein expression in infected cells.     

Studies on a fractionated murine fibrosarcoma: A reproducible method for the cautious and a caution for the unwary.

A technique is described for the dissociation and fractionation by isopycnic centrifugation (4,000 × g, 60 minutes, 4°C) in linear bovine albumin density gradients (12 ml, pH 5.2 real osmolality 333 mmol/Kg water, 1.030–1.075 g/cm³) of cells (? 3 × 10⁷/gradient) released by a strictly standardised combination of mechanical and enzymatic means from a transplantable methylcholanthrene induced BALB/c fibrosarcoma. Optimal conditions for reproducible localisation of cell bands with maintenance of both satisfactory resolution and satisfactory viable cell recovery (> 80%) were established by means of a series of simultaneous double fractionation experiments. When rebanding was performed under these conditions the density of the median of the cell count of the refractioned cells shifted less than 0.0005 g/cm³. Experiments also showed that close adherence to certain aspects of the tissue dissociation and fractionation protocol was necessary to avoid reduced cell yields and viabilities, density-dependent selective cell losses, reductions in resolution and shifts in the location of cell bands. Other aspects of the protocol were tolerant to variation without introducing artefacts.