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Long-term, large scale cryopreservation of insect cells at -80 °C.

ABSTRACT: Standard tissue culture methods advise freezing cells in small aliquots (?1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10(8) insect cells at -80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were "expression ready" immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at -80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.

SUBMITTER: Vyletova L

PROVIDER: S-EPMC4754242 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Long-term, large scale cryopreservation of insect cells at -80 °C.

Vyletova Lucie L Rennalls La'Verne P LP Wood Kirstin J L KJ Good Valerie M VM

Cytotechnology 20140823 2

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the lo ...[more]

PMID: 25149288

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Project description:BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Project description:Climate is the predominant environmental driver of freshwater assemblage pattern on large spatial scales, and traits of freshwater organisms have shown considerable potential to identify impacts of climate change. Although several studies suggest traits that may indicate vulnerability to climate change, the empirical relationship between freshwater assemblage trait composition and climate has been rarely examined on large scales. We compared the responses of the assumed climate-associated traits from six grouping features to 35 bioclimatic indices (~18 km resolution) for five insect orders (Diptera, Ephemeroptera, Odonata, Plecoptera and Trichoptera), evaluated their potential for changing distribution pattern under future climate change and identified the most influential bioclimatic indices. The data comprised 782 species and 395 genera sampled in 4,752 stream sites during 2006 and 2007 in Germany (~357,000 km² spatial extent). We quantified the variability and spatial autocorrelation in the traits and orders that are associated with the combined and individual bioclimatic indices. Traits of temperature preference grouping feature that are the products of several other underlying climate-associated traits, and the insect order Ephemeroptera exhibited the strongest response to the bioclimatic indices as well as the highest potential for changing distribution pattern. Regarding individual traits, insects in general and ephemeropterans preferring very cold temperature showed the highest response, and the insects preferring cold and trichopterans preferring moderate temperature showed the highest potential for changing distribution. We showed that the seasonal radiation and moisture are the most influential bioclimatic aspects, and thus changes in these aspects may affect the most responsive traits and orders and drive a change in their spatial distribution pattern. Our findings support the development of trait-based metrics to predict and detect climate-related changes of freshwater assemblages.

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Long-term, large scale cryopreservation of insect cells at -80 °C.

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Long-term, large scale cryopreservation of insect cells at -80 °C.

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