Protectants used in the cryopreservation of microorganisms

Hubálek, Zdeněk

doi:10.1016/s0011-2240(03)00046-4

Cited by 645 publications

(526 citation statements)

References 206 publications

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“…DMSO and glycerol are the most common protectants added to bacterial cells to enhance cryopreservation (Hubalek, 2003). However, low concentrations of DMSO might result in cellular toxicity (Galvao et al ., 2014), which limits its use for FMT unless a removal step is used before transplantation.…”

Section: Discussionmentioning

confidence: 99%

“…However, ice formation during freezing can cause lethal damage to bacterial cells; thus, cryoprotectants must be added to prevent cryoinjuries. The positive effects of different protective matrices composed out of polysaccharides, amino acids, peptides or more complex compounds on microbial cell physiology during freezing, storage and thawing have been studied for pure cultures and are well approved (Hubalek, 2003). Non‐penetrating cryoprotectants, such as many saccharides, reduce ice formation within cells by osmosis‐derived dehydration before freezing.…”

Section: Introductionmentioning

confidence: 99%

“…Cryoprotective sugars can also bind to the cell surface and inhibit extracellular ice crystal formation. Penetrating cryoprotectants, such as glycerol and dimethyl sulfoxide (DMSO), change fluid properties and increase membrane glass‐phase transition temperature, which results in reduced intracellular ice formation (Hubalek, 2003; Fowler and Toner, 2005). Nevertheless, DMSO is not recommended for in vivo administrations as it was found to be toxic to cells at low concentrations of 2–4% (Galvao et al ., 2014), whereas glycerol is a common additive for frozen faecal samples also in stool banks (Aguirre et al ., 2015).…”

Section: Introductionmentioning

confidence: 99%

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Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Bircher

Schwab

Geirnaert

et al. 2017

Microbial Biotechnology

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SummaryInterest in faecal microbiota transplantation (FMT) has increased as therapy for intestinal diseases, but safety issues limit its widespread use. Intestinal fermentation technology (IFT) can produce controlled, diverse and metabolically active ‘artificial’ colonic microbiota as potential alternative to common FMT. However, suitable processing technology to store this artificial microbiota is lacking. In this study, we evaluated the impact of the two cryoprotectives, glycerol (15% v/v) and inulin (5% w/v) alone and in combination, in preserving short‐chain fatty acid formation and recovery of major butyrate‐producing bacteria in three artificial microbiota during cryopreservation for 3 months at −80°C. After 24 h anaerobic fermentation of the preserved microbiota, butyrate and propionate production were maintained when glycerol was used as cryoprotectant, while acetate and butyrate were formed more rapidly with glycerol in combination with inulin. Glycerol supported cryopreservation of the Roseburia spp./Eubacterium rectale group, while inulin improved the recovery of Faecalibacterium prausnitzii. Eubacterium hallii growth was affected minimally by cryopreservation. Our data indicate that butyrate producers, which are key organisms for gut health, can be well preserved with glycerol and inulin during frozen storage. This is of high importance if artificially produced colonic microbiota is considered for therapeutic purposes.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Bircher

Schwab

Geirnaert

et al. 2017

Microbial Biotechnology

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“…In addition, their suitability to longer-term storage protocols is doubtful. Another largely used technique is cryopreservation from -60 to -130°C in ultrafreezers or at -196°C with liquid nitrogen [6], both considered the best way to keep cell viability. However, the preservation at -196°C often requires substantial initial outlay, and an alternative method without the use of costly equipment would provide significant benefits mainly for filamentous fungus maintenance.…”

Section: Introductionmentioning

confidence: 99%

“…One of the main cell injuries to overcome in cryopreservation at -20°C is ice formation in the cell interstice [8] which causes mechanical injuries [7]. An alternative to reduce these cryoinjuries is the use of cryoprotective agents [6] that decrease ice formation and/or affect the plasmatic membrane elasticity reducing cellular breaking and increasing mycelial viability [9]. Another alternative to reduce cryoinjuries is the use of different substrates for mycelial growth before freezing.…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

et al. 2012

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Alternative substrates for cryopreservation at -20°C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between -15 and -60°C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at -20 or -70°C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at -20 or -70°C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at -20 or -70°C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at -20°C for 1 or 3 years; for cryopreservation at -70°C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at -20 or -70°C for at least 3 years.

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Cryopreservation of Plant Cells

Keller¹,

Senula²,

Höfer³

et al. 2013

Encyclopedia of Industrial Biotechnology

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Cryopreservation, storage of living tissues in or above liquid nitrogen, is increasingly used to preserve germplasm in the long term. When cooling plant tissue to low temperatures, water may destroy cells by osmotic drying or disruption of organelles by forming ice crystals. The organisms accumulate cryoprotective substances to adapt to freezing. Cryoprotectants trigger glass transition (vitrification), when temperature is cooled down rapidly. In the course of cryopreservation, tissues are imposed by various influences causing oxidative stress: wounding, dehydration, and direct liquid nitrogen effect. Occurring reactive oxygen species may be measured and antioxidant substances may be added. Cell structure alterations, water status, regeneration from cryopreservation injury, and genetic and epigenetic changes, which may occur, were investigated. Various plant tissues were cryopreserved: dedifferentiated cells and tissues; shoot tips; axillary buds and other meristematic plant parts; dormant buds of woods species, seeds, embryos, and embryo axes; and pollen. Several main methods were used: slow freezing, vitrification, and encapsulation. Liquid nitrogen can also be used to eliminate viruses from the organs (cryotherapy). The existence of endogenous or contaminating microorganisms needs special attention. Furthermore, technological considerations and the use of cryopreservation to maintain transgenic or patented material need to be mentioned.

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Protectants used in the cryopreservation of microorganisms

Cited by 645 publications

References 206 publications

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Cryopreservation of Plant Cells

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