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991.
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993.
994.
Alvin W. Nienow 《Cytotechnology》2006,50(1-3):9-33
This article mainly addresses the issues associated with the engineering of large-scale free suspension culture in agitated bioreactors >10,000 L because they have become the system of choice industrially. It is particularly concerned with problems that become increasingly important as the scale increases. However, very few papers have been written that are actually based on such large-scale studies and the few that do rarely address any of the issues quantitatively. Hence, it is necessary very often to extrapolate from small-scale work and this review tries to pull the two types of study together. It is shown that ‘shear sensitivity’ due to agitation and bursting bubbles is no longer considered a major problem. Homogeneity becomes increasingly important with respect to pH and nutrients at the largest scale and sub-surface feeding is recommended despite ‘cleaning in place’ concerns. There are still major questions with cell retention/recycle systems at these scales, either because of fouling, of capacity or of potential and different ‘shear sensitivity’ questions. Fed-batch operation gives rise to cell densities that have led to the use of oxygen and enriched air to meet oxygen demands. This strategy, in turn, gives rise to a CO2 evolution rate that impacts on pH control, pCO2 and osmolality. These interactions are difficult to resolve but if higher sparge and agitation intensities could be used to achieve the necessary oxygen transfer, the problem would largely disappear. Thus, the perception of ‘shear sensitivity’ is still impacting on the development of animal cell culture at the commercial scale. Microcarrier culture is also briefly addressed. Finally, some recommendations for bioreactor configuration and operating strategy are given. 相似文献
995.
Summary By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene. When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC
+
and recF
+
genes. When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis. This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative. Thus, we conclude that the induced (unuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers. glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9. This uninduced mutagenesis is independent of the umuC
+
, recF
+
and recA
+
genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides. The role of recF in UV mutagenesis was tested in two ways. First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent. Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play. Second, UV mutagenesis of E. coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent. We conclude that the mechanism of induced UV mutagenesis of the E. coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference. 相似文献
996.
Elena Costariol Marco Rotondi Arman Amini Christopher J. Hewitt Alvin W. Nienow Thomas R.J. Heathman Martina Micheletti Qasim A. Rafiq 《Biotechnology and bioengineering》2019,116(10):2488-2502
Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags. In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg−1), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 106 viable cells ml−1 obtained at the highest P/M of 74 × 10−4 W kg−1 in comparison with 0.91 ± 0.07 × 106 viable cells ml−1 at the lowest P/M of 3.1 × 10−4 W kg−1. We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications. 相似文献
997.
Stilwell Justin M. Petty B. Denise Camus Alvin C. Woodyard Ethan T. Griffin Matt J. Rosser Thomas G. 《Systematic parasitology》2020,97(3):305-314
Systematic Parasitology - A previously undescribed Myxobolus sp. was isolated from the cranial nerves and ganglia of the spotfin hatchetfish Thoracocharax stellatus (Kner) that exhibited neurologic... 相似文献
998.
Alvin F. Reeves II 《American journal of botany》1977,64(2):186-189
A new trichome type for the genus Lycopersicon is described in L. esculentum Mill. It is a short (0.03–0.08 mm), pendant, glandular hair with a club-shaped head consisting of 8–12 cells. Two previously described “hairless” mutations were examined microscopically. One, hl, does not affect the frequency of hairs nor the number of cells per hair, but causes abnormal enlargement of the stalk cells of all hair types, and thus produces shortened, extremely bent and twisted hairs. Observations on the time of action of this gene indicate that in trichome development two to four cell divisions occur prior to any appreciable cell enlargement. The second mutation, h, affects only the large type of trichome. This mutation effects a developmental shift from trichome to stomatal apparatus at the apex of the multicellular base normally supporting the large trichomes. 相似文献
999.
Summary Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4–6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiological events associated with voltage clamping (V
t
=inside-outside potential). Increase and decrease ofV
t
produced converse effects, related directly to the magnitude ofV
t
.Hyperpolarization resulted in prompt decrease in inward transepithelial currentI
t and increase in fractional outer membrane resistancefR
0 (as evaluated from small transient voltage perturbations) and in outer membrane potentialV
0. Overshoot ofV
0 was followed by relaxation to a quasi-steady state in minutes. Changes infR
0 were progressive, with half times of some 1–5 sec. Changes in transepithelial slope conductanceg
t
were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductancesg
0 andg
i
. Nevertheless, it was shown thatg
0 is related inversely toV
t
andV
0. Presuming insensitivity ofV
i
toV
t
, the dependence ofg
0 onV
0 in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence ofg
0 onV
0 and/or cellular current. In contrast to previous findings in tight epithelia at open circuit, differences inV
t
were associated with substantial differences infR
0 and inner membrane potentialV
i
. Hyperpolarization ofV
t
over ranges commonly employed in studies of active transport and metabolism appears to increase significantly the electrochemical work per Na ion transported. 相似文献
1000.