BENEFICIAL EFFECTS OF PLURONIC F-68 AND ARTIFICIAL OXYGEN CARRIERS ON THE POST-THAW RECOVERY OF CRYOPRESERVED PLANT CELLS

Abstract

The storage of prokaryotic and eukaryotic cells at ultra-low temperature in liquid nitrogen (−196°C) is a procedure that has assumed an increasingly important role in underpinning many aspects of biotechnology. For eukaryotic cells, the transition from a cryopreserved state to physiologically normal temperatures and oxygen tensions, induces respiratory imbalances that may stimulate the production of toxic oxygen radicals causing impaired cellular functions. Novel treatments, that focus specifically on enhancing oxygen delivery to cells, are important in maximising post-thaw recovery. Recently, several approaches have been evaluated with suspension cultured plant cells as a model, yet biotechnologicallyimportant, totipotent eukaryotic cell system. Such treatments include non-ionic surfactants, primarily Pluronic F-68, and artificial oxygen carriers, the latter based on inert perfluorochemical liquids or chemically-modifed haemoglobin, as supplements to culture medium used during the post-thaw recovery phase of cell growth. When used either alone or in combination, such novel treatments stimulate significantly the post-thaw viability and biomass production of cultured plant cells. Many of these technologies will be exploitable in cryopreservation protocols for eukaryotic cells in general.

CRYOPRESERVATION OF PLANT CELL SUSPENSION CULTURES

De-differentiated plant cells maintained as suspension cultures in liquid medium have been cryopreserved and recovered in several agronomically-important crops, including artichoke [[1]], asparagus [[2]], banana [[3]], barley [[4]], blackberry [[5]], grape [[6]], mandarin and mandarin hybrids [[7]], millet [[8]], pine [[9]], rice [[10]], rubber [[11]] and soybean [[12]].

Embryogenic cell suspensions are used routinely as source material for the preparation of totipotent protoplasts (wall-less cells) for plant genetic manipulation, particularly in the case of cereals, such as rice [[13]]. Such embryogenic cell cultures also provide an alternative to zygotic embryo-derived tissues for the production of fertile, transgenic rice plants following gene delivery by biolistics [[14]]. Embryogenic suspension-cultured cells are also amenable to Agrobacterium-mediated transformation [15-16]. In general, the establishment and maintenance of embryogenic cell suspensions of rice, especially Indica rices, presents technical difficulties, since the morphogenic competence of suspensions declines with subculture [[17]]. However, such loss of totipotency is not unique to rice suspension cultures. Additionally, during the period when plant regeneration does occur, the plants are often morphologically abnormal and infertile [[18]].

Cryopreservation is now exploited routinely for the long-term storage of biological materials at ultra-low temperatures [[19]]. This technique circumvents the loss of totipotency in plant systems and eliminates or reduces genetic perturbations, together with alterations in secondary product biosynthesis in some systems, frequently associated with extended culture of suspensions at physiologically normal temperatures (ca. 25°C). Cryopreservation also negates the labour-intensive requirement to re-initiate periodically and to characterise new cell lines in terms of their growth and totipotency. Moreover, storage at ultra-low temperature provides a readily available and constant supply of morphogenetically competent cells [[20]]. Some of the advantages and limitations of cryopreservation are listed in Table 1.

Table I. Some Advantages and Disadvantages of the Cryopreservation of Cells

Advantages Storage and conservation of elite and endangered germplasms Facilitates germplasm transportation and distribution

Disadvantages Not applicable to all cell types Dependant on specific pre- and post-thaw treatments Cell loss on thawing Post-thaw physiological changes

PHYSICAL AND CHEMICAL PARAMETERS FOR STIMULATING POST-THAW RECOVERY OF CELLS

Early cryopreservation studies with rice cell suspensions involved assessments of the freezing and thawing conditions that permit the recovery of viable cells and their subsequent growth in culture [[20]]. Previously, Meijer et al. [[21]] reported that supporting post-thawed rice cells on filter paper disks, as opposed to placing cells directly on the surface of the culture medium employed during cell recovery, resulted in significant increases in cell regrowth. Subsequently, Lynch et al. [[20]] also found that the duration of post-thaw culture was important prior to the transfer to new medium of filter disks supporting the thawed cells, with the correct timing of the transfer decreasing the period required to re-establish cell suspensions. Additionally, these workers noted that the recovery of rice cells from freezing in 9 cm diameter Petri dishes was superior compared to their culture in 5.5 cm diameter vessels. Presumably, in the larger dishes, the greater volume of culture medium assisted the rehydration of cells and the diffusion from the cells of the potentially toxic cryoprotectant, dimethyl sulphoxide (DMSO), together with any cell oxidation products. Interestingly, the inclusion of activated charcoal in the cell recovery medium has beneficial effects on the post-thaw recovery of suspension cultured cells of grape [[22]], presumably through the absorption of toxic cellular waste products.

RESPIRATORY IMBALANCES DURING POST-THAW RECOVERY OF CRYOPRESERVED CELLS

Several distinct stages are involved in cryopreservation, of which freezing is one (Table 2). The successful and reproducible recovery of frozen cells depends upon the pre-freezing, cryogenic and post-freezing conditions. The transition of cells from ultra-low temperature (−196°C) in liquid nitrogen to physiologically normal temperatures and oxygen tensions, induces respiratory imbalances which stimulate the production of toxic oxygen radicals [[23]]. Interestingly, in this respect, physiological investigations of cryopreserved cell suspension cultures of rice have already demonstrated respiratory impairment associated with post-thaw recovery of such cells [[24]]. Furthermore, in the case of barley, it was observed that pre-treatment of embryogenic cell suspensions for 3 days with high concentrations (29–87 mM) of the antioxidant ascorbic acid (vitamin C) prior to freezing, had a positive effect on post-thaw oxidative stress and lipid peroxidation through the scavenging of oxygen-free radicals [[4]]. It is also well established that treatment of cells with transition metals, particularly iron, during the early stages of post-thaw cell recovery, catalyses the production of active oxygen intermediates. The latter react with the polyunsaturated fatty acids in tissue membranes leading to lipid peroxidation [[25]]. The same workers investigated the effects of supplementing the post-thaw recovery medium of rice cells with the iron chelating drug, desferrioxamine, which had been used previously to reduce oxidative stress in mammalian tissues subjected to low temperatures [[26]]. However, although positive and beneficial effects were observed in relation to cell viability and cellular regrowth of rice cells, lipid peroxidation was not significantly reduced as assessed by thiobarbituric acid (TBA) production [[25]]. More recently, Fleck et al. [[27]] demonstrated that desferrioxamine similarly improved the post-thaw survival of the green alga, Euglena gracilis. This latter study represents an elegant example of the application of exogenous anti-oxidant treatments developed for medical applications being exploited in cryopreservation protocols for plant cells.

Table II. A Typical Cryopreservation Protocol for Eukaryotic Cells

Pre-freeze treatment(s) Addition of cryoprotectant (e.g. dimethyl sulphoxide, glycerol) Freezing in liquid nitrogen ♦ ‘flash’ immersion ♦ programmed freezing Cryostorage Thawing Post-thaw treatment(s)

NOVEL POST-THAW TREATMENTS TO ENHANCE OXYGEN DELIVERY TO THAWED CELLS

Novel treatments that enhance oxygen delivery to eukaryotic cells, are important in maximising their post-thaw recovery. Indeed, several approaches have been evaluated, including the use of non-ionic surfactants, exposure to inert, respiratory-gas dissolving perfluorochemical liquids and chemically-modified haemoglobin. Such compounds have been used as supplements to the culture medium during the post-thaw recovery phase of cell growth. Cell suspensions of rice have been exploited as a “model” totipotent system in several of these investigations, because of the importance of rice as a major cereal crop and the considerable information in relation to its cytology and genomic constitution.

Supplementation of Culture Medium with the Surfactant Pluronic® F-68

Pluronic® F-68 (Poloxamer 188; Table 3), a non-ionic, polyoxyethylene (POE)-polyoxypropylene (POP) block co-polymer surfactant, has been employed extensively as a non-toxic, low cost, cell-protecting agent during the culture of both animal [28-33] and plant [34-35] cells. The surfactant is believed to protect cells against fluid-mechanical damage by acting as a foam-stabilising agent in agitated/aerated cultures [[31]], [[33]], [36-38]. Indeed, a recent theoretical study, that examined cell attachment to rising bubbles in sparged bioreactors, has confirmed that Pluronic F-68 at 0.1% (w/v) inhibits such cell-bubble attachment [[39]]. Studies with cultured animal cells have demonstrated that Pluronic® F-68 is adsorbed onto cell surfaces [[29]], [[31]], [34-35], while investigations with cultured insect cells [[31]], [40-41] and mammalian hybridomas [[42]] have shown that interaction of Pluronic® F-68 with cytoplasmic membranes increases the resistance of the treated cells to shear forces, albeit over different periods of time, depending on the cell type. The Pluronic polyols have hydrophobic POP cores; the latter are believed to become embedded in the phospholipid membranes of cells, leaving their hydrophilic, POE tails outside. This has the effect of reducing the interfacial tension of the cells and sterically hindering adhesive interaction between molecules on the cell surfaces. Such reduction in adhesive interaction prevents cell-to-cell contact which, thus, further reduces mechanical damage.

Table III. General Chemical and Physical Properties of Pluronic® F-68

Average values for a, b and c = 75, 30 and 75, respectively.
Mean molecular weight	8350
Melting point (°C)	50
Physical form at 20°C	solid
Cloud point (°C)^*	>100
HLB^**	29

^* Cloud point = temperature at which solutions of non-ionic surfactants become cloudy and undergo phase separation to yield a detergent-rich layer and an aqueous layer.

^** HLB = hydrophilic-lipophilic balance number, an empirical measure of the emulsifying power of a surfactant. Compounds with low HLB values (<20) are hydrophobic, whereas those with higher values (>20) are hydrophilic.

Data from References [43-44].

Initial studies, performed more than four decades ago, showed that the Pluronics could prevent haemolysis of human red blood cells in response to freeze-thawing procedures [[45]]. Subsequently, experiments demonstrated that Pluronic® F-68 was effective in cryoprotecting cultured Chinese Hamster cells [[46]]. Surprisingly, the effects had not been evaluated of exposing plant cells to Pluronic® F-68, either as a cryoprotectant per se, or as a post-thaw cytoprotecting agent, inspite of the increasing interest in cryopreservation for conserving agronomically-important and endangered plant germplasms [[47]]. Consequently, the cytoprotectant properties of Pluronic® F-68 together with the cryoprotectant effects of Pluronics in animal cell systems, made this compound an obvious candidate for evaluation and possible inclusion in protocols for the cryopreservation of plant cells. Therefore, investigations with embryogenic suspension cultured cells of the Japonica rices, Oryza sativa cvs. Taipei 309 and Tarom, together with nonembryogenic cells of Lolium multiflorum (Italian ryegrass), were initiated to evaluate the potential beneficial effects of Pluronic® F-68 at 0.01–0.2 % (w/v). The surfactant was employed either as a cell-protectant during freezing, or as a specific culture medium supplement to enhance the post-thaw cellular growth following short-term (30 days; O. sativa cv. Tarom, Lolium multiflorum) or longterm (3 years; O. sativa cv. Taipei 309) cryopreservation of cells at −196°C in liquid nitrogen [[48]].

Assessments of the post-thaw growth of rice and ryegrass cells involved studies of biomass production and the reduction of triphenyl tetrazolium chloride (TTC) [[49]]. The TTC assay is used routinely in post-thaw protocols to assess the metabolism of tissues by their ability to reduce enzymatically TTC to red formazan; the latter can be quantified spectrophotometrically. Assessment of TTC reduction was made at 4 days after thawing of rice and ryegrass cells, since previous studies indicated that the TTC assay, at this time, provided optimal information on cell metabolic capacity [[20]].

A significant finding in these experiments was that, following TTC reduction, the mean absorbance of Taipei 309 cells 4 days after thawing was increased 2-fold over the control value by 0.01% (w/v) of Pluronic® F-68 and almost 2-fold by the surfactant at 0.1% (w/v). A more pronounced, 4-fold increase (P < 0.001) in post-thaw absorbance occurred with Tarom cells exposed to 0.1% (w/v) Pluronic® F-68; cell absorbance was elevated 3-fold with 0.2% (w/v) Pluronic® F-68 (P < 0.001), but only 2-fold (P < 0.01) by 0.01% (w/v) of the surfactant. In cells of Lolium, TTC reduction was elevated by 31% (P < 0.01) when cells were recovered in the presence of 0.01% (w/v) of the surfactant following thawing. The presence of Pluronic® F-68 at 0.01% (w/v) also promoted sustained mitotic activity, since biomass, measured 30 days post-thawing, was increased to a maximum of 32% above the control value (P < 0.05) in cells of both Taipei 309 and L. multiflorum. Interestingly, there was no measurable beneficial effect of supplementing the cryoprotectant with Pluronic® F-68 before cells of the rice cvs. Taipei 309 and Tarom and ryegrass were frozen for 14 days. In these cases, the cryoprotectant consisted of a mixture of 0.5 M glycerol, 0.5 M dimethyl sulphoxide (DMSO), 1.0 M sucrose and 0.04 M proline prepared in the culture medium appropriate for rice or ryegrass. These results, which demonstrated a marked cytoprotection of Pluronic® F-68 on plant cells recovered from cryo-storage, suggested that this surfactant was a useful additive during post-thaw handling strategies.

Overall, Pluronic® F-68 increased the post-thaw metabolism and growth of cryopreserved rice and ryegrass cells. It is generally accepted that reduction of TTC can be used as an indicator of cell viability and metabolism [[25]]; the investigation by Anthony et al. [[48]] also included data on biomass increases to support the conclusion of the growth-enhancing effects of Pluronic® F-68. It is probable that the observations reflected a combination of increased cell survival following thawing, perhaps coupled with a growth-stimulating effect of the surfactant as reported previously for cultured plant cells [[34]].

A noteworthy observation from the study of Anthony et al. [[48]] was that the optimum concentration of surfactant, which increased cell growth, differed between the two rice cultivars and between the rice cv. Tarom and the nonembryogenic suspension cultured cells of ryegrass. Thus, there was evidence for both species and cultivar-specific responses to Pluronic® F-68. Such responses of cryopreserved cells were consistent with previous observations with plant cells and tissues that had not been exposed to ultra-low temperatures. For example, in Chrysanthemum morifolium, the optimum concentration of Pluronic® F-68 that stimulated adventive shoot regeneration from cultured leaf explants differed by an order of magnitude between cultivars [[50]]. Differences in the responsiveness of tissues and organs to the growth-promoting effects of Pluronic® F-68 have also been observed in Solanum dulcamara [[51]], Corchorus capsularis [[52]], Hypericum perforatum [[53]], Populus spp. [[54]] and Manihot esculenta [[55]].

The effect of Pluronic® F-68 on plant cells may be to promote the uptake of nutrients, growth regulators or oxygen into cells during the post-thaw culture period [[34]]. Similar experiments with chick fibroblasts cultured under static conditions have demonstrated that Pluronic® F-68, at concentrations comparable to those used in the investigation with rice and ryegrass, stimulated both 2-deoxyglucose uptake and cellular amino acid incorporation [[56]]. Any increase in nutrient uptake promoted by Pluronic would be expected to alter metabolic flux, allowing biochemical pathways to operate more efficiently, particularly under the stress conditions of early post-thaw recovery. Indeed, previous studies have shown that respiratory impairment occurs during the early post-thaw period of cryopreserved rice suspension cells [24-25]. Consequently, it is possible that Pluronic® F-68 assists in overcoming such perturbations. The adsorption of Pluronic molecules onto the cytoplasmic membranes of post-thawed plant cells may also reduce any cellular damage that occurs during rehydration that accompanies the progressive removal of DMSO from the cryoprotectant [[57]].

More extensive studies are essential to determine the mechanism(s) by which surfactants, such as Pluronic® F-68, improve the growth and survival of plant cells after thawing. A focus of such work should be to correlate the effects of Pluronic® F-68 with respiratory gas dynamics, since this surfactant can alter oxygen transport in agitated, sparred bioreactors [[58]]. Additionally, Pluronic® F-68 may influence carbon dioxide release from cells (34 or inhibit ethylene production in a manner comparable to that promoted by Triton X-100 [[59]].

The results available at present, although limited, imply that Pluronic® F-68, which is freely available commercially (e.g. from Sigma Chemical Co., Poole, UK) and relatively inexpensive, could be a routine supplement of plant culture media in order to increase cell recovery and growth during post-thaw handling procedures.

Perfluorochemical Liquids to Underlay Cell Culture Media

The optimisation of the post-thaw (re-growth) culture conditions, including the regulation of respiratory gases, is essential in order to maximise the recovery from freezing of cryopreserved plant cells. A novel approach for enhancing the oxygen supply to post-thawed cryopreserved cells is the use of chemically-inert, perfluorochemical (PFC) liquids. PFCs are linear, cyclic or bicyclic hydrocarbons in which most or all of the hydrogen atoms have been replaced with fluorine. They are colourless, odourless liquids that have specific gravities about twice that of water; the applications and benefits of PFCs to the culture of plant and microbial cells have been reviewed [[60]] and are summarised in Table 4. A significant attribute of PFC liquids is that they dissolve substantial volumes of oxygen and other respiratory gases. For example, typical values for the solubility of oxygen and carbon dioxide in liquid PFCs are 35–44 mmol l ¹ and 123–200 mmol ¹ l, respectively. PFCs have been studied in, for example, emulsified form as vehicles for oxygen transport in vivo [61-64]. The basis for exploiting PFC liquids in recovering cells from cryopreservation, relates to their use to enhance oxygen supply to cultured protoplasts and protoplast-derived cells of a range of plants, including those of cassava (Manihot esculenta), passion fruit (Passiflora edulis; P. giberti) and rice, together with the ornamentals petunia (Petunia hybrida; P. parodii) and Salpiglossis sinuata at temperatures (about 25°C) normally employed in standard tissue culture protocols [65-68].

Table IV. Benefits of Using PFCs in Cell Culture Systems

Chemically and biologically inert Easily sterilised (e.g. by autoclaving) Recoverable and recycleable High respiratory gas solubility Scavengers of gaseous cellular products (e.g. ethylene) Provide a two-phase (PFC liquid–aqueous medium) interface and physical support system

From Reference [[60]].

Embryogenic (totipotent) suspension cells of rice, capable of regenerating plants by somatic embryogenesis, have been used to assess the potential beneficial effects of oxygenated perfluorodecalin (C₁₀F₁₈; Flutec® PP6; F2 Fluorochemicals Ltd., Preston, UK) on the post-thaw viability, following long-term cryopreservation in liquid nitrogen (ca. 3 years), of suspension cultured cells of the rice cv. Taipei 309. In these experiments, thawed cells were placed on two superimposed 2.5 cm diameter Whatman No. 1 filter paper disks overlaying 5.0 ml aliquots of semi-solidified culture medium. The latter itself had previously been overlaid onto 20 ml aliquots of oxygenated (10 mbar, 15 min) perfluorodecalin. The recovery of cells was performed in 100 ml capacity screw-capped glass jars. The postcryopreservation handling protocol was further modified, using 24 well Petri dishes, to evaluate the feasibility of recovering the cells on a smaller scale, in order to reduce the volume of perfluorodecalin employed. Each well contained 2.0 ml of oxygenated perfluorodecalin over which was placed 1.0 ml of semi-solidified culture medium, the latter being overlaid with two 1.3 cm diameter filter papers. The mean cell viability, as assessed by TTC reduction at 4 days after thawing, was increased by 20% over the control value by oxygenated perfluorodecalin (P < 0.05) in 100 ml glass jars and, similarly, by 24% (P < 0.01) when the cells were recovered on a smaller scale in 24 well Petri dishes. Studies also conducted using protoplasts isolated from unfrozen cell suspensions of the rice cv. Taipei 309 showed that mitotic division was enhanced during culture of the protoplasts at the interface between oxygen-gassed PFC overlaid with liquid or agarose-solidified culture medium. Shoot formation was also stimulated in this totipotent cell system [[69]]. This information confirmed a genuine growth enhancement of PFC treatment, rather than a marginal effect on cells recovered from cryopreservation.

It is believed that PFC liquids act as reservoirs for oxygen that diffuses into the aqueous medium/cell phase during the initial culture of protoplasts, this being supported by changes in oxygen tension in the medium [[70]]. It is likely that the increased and sustained post-thaw growth of rice cells was also due to an enhanced oxygen supply provided by the PFC. Indirect evidence for the diffusion of oxygen from the PFC into the aqueous culture medium is also provided by related observations with cell suspension-derived protoplasts of Salpiglossis sinuata, in which an increase in intracellular superoxide dismutase (SOD) occurred after 3 days of culture in medium overlaying oxygen-gassed PFC [[71]]. These observations, in turn, were consistent with studies using Mycobacterium spp., in which SOD activity was increased during culture of the bacteria in perfluorodecalin supplemented medium [[72]].

Lipid peroxidation and protein degradation occur during the early post-thaw recovery stages of plant cells [[23]], [[26]], leading to the production of toxic oxygen radicals. An increase in SOD biosynthesis during culture of cells in the presence of oxygenated PFC may protect the cells not only against a supplemented oxygen supply, but also against oxygen radicals generated by impaired oxygen flux during thawing. However, additional investigations are needed to clarify the early timecourse of changes in SOD and other oxygen-sensitive enzymes during the recovery of cells from cryopreservation.

PFCs may also be useful as gas delivery vehicles for cryopreserved, multicellular explants in the context of cryopreservation. For example, the conversion to intact plants of thawed apical meristems may be facilitated by oxygen-gassed PFC. Thus, the latter can be used to promote initial post-cryopreservation survival, followed by exposure of meristem-derived shoots to carbon dioxide-gassed PFC to stimulate rooting and ex vitro acclimation [[73]]. Related studies in animal systems have shown that PFCs and their emulsions facilitate survival of fish semen during low temperature storage [[74]] and promoted hypothermic preservation of mammalian organs [[75]].

Synergistic Effects of Surfactants and PFC

Surfactants and PFCs may exert a synergistic effect. Thus, studies with animal cells have demonstrated that PFC, emulsified with Pluronic® F-68, prolonged the fertilising capability of turkey spermatozoa stored at 4°C [[76]]. Whilst specific effects of Pluronic were not evaluated, the surfactant may have contributed to cell survival, either through cytoprotection or possibly, by enhancing oxygen and/or nutrient uptake. In this respect, a recent study has evaluated the effectiveness of the related surfactant, Pluronic F-127, alongside other cell protecting agents (e.g. egg yolk phospholipids, glycerol) for enhancing the post-thaw functions of cryopreserved boar sperm [[77]]. It was noted that sperm motility and acrosome morphology were improved in the presence of Pluronic F-127, further highlighting the potential routine use of Pluronic polyols in sperm cryopreservation.

In evaluating any synergistic effects of PFC and surfactants in plant systems, PFC either alone and in combination with 0.01 (w/v) Pluronic® F-68 was used to supplement the medium used to recover cryopreserved cells of the rice cultivar, Taipei 309. Indeed, the results of such experiments demonstrated a synergistic effect of such compounds. Thus, a 21% increase in post-thaw viability above the values for the control treatments (P < 0.05) was observed with oxygenated perfluorodecalin alone and a 36% increase (P < 0.05) in post-thaw viability when cells were exposed to medium supplemented with only 0.01% (w/v) Pluronic® F-68. However, a more pronounced, synergistic increase in viability of up to 57% over control (P < 0.05) was recorded when cryopreserved rice cells were recovered with both oxygenated perfluorodecalin and 0.01% (w/v) Pluronic® F-68 [[48]]. Similarly, perfluorodecalin and Pluronic® F-68 treatments, used either alone or in combination, also promoted an increase in biomass, when measured as fresh weight gain 30 days after thawing of rice cells. This increase was to a maximum of 38% above the control value (P < 0.05) in the case of PFC alone and when oxygen-gassed perfluorodecalin was used in combination with Pluronic® F-68. Significantly, when the PFC and Pluronic® F-68 treatments were combined, the increase in biomass was greater than that of cells recovered in the presence of Pluronic® F-68 alone, but not significantly different from that of PFC alone.

Currently, the results with plant cells indicate that oxygenated PFC underlying the culture medium, in combination with a surfactant such as Pluronic® F-68 in the medium itself, may be useful in maximising plant cell viability during culture after recovery from cryopreservation. However, once cells have recovered from the freezing process, there is no additional advantage of retaining both oxygen-gassed PFC and surfactant in the culture medium, since the presence of oxygen-gassed PFC alone is adequate to maximise biomass production. Certainly, this is the case in rice cells. The use of PFC and surfactant options may be especially applicable to post-thaw handling strategies for cells of those plants which, normally, respond poorly to conventional recovery procedures after storage in liquid nitrogen. Future studies should confirm the value of such post-thaw treatments. An additional advantage of using PFCs in plant cryopreservation protocols is that they are easy to recover and recycle, thereby providing a cost effective underpin to germplasm storage technologies [[60]], [[67]]. The fact that PFCs can be exploited in small-scale culture systems, makes their routine use economically feasible.

Supplementation of Culture Medium with Haemoglobin (Erythrogen™)

Considerable effort has focused on assessments of native and, to a greater extent, chemically-modified haemoglobins as vehicles for respiratory gas transport in animal cell systems [63-64], [78-81]. The most widely studied molecules are of human or bovine origins, although recombinant haemoglobins, expressed in Escherichia coli [[82]] and transgenic mice [[83]] or pigs [[84]], have also been reported. An innovation has been the expression of bacterial haemoglobin genes in transgenic tobacco [85-86]. It will be of interest to evaluate whether the impaired oxygen flux, which is characteristic of many plant cells recovered from cryopreservation, is similar in transformed plant cells expressing haemoglobin transgenes.

Normal haemoglobin is composed of a protein component (globin) made up of four polypeptide chains, each associated with a porphyrin derivative (haem) containing one atom of iron in the ferrous state (Fe²⁺). Each iron atom can rapidly and reversibly bind with one oxygen molecule, although the reaction is oxygenation rather than oxidation, as the redox status of the iron remains unchanged. Since the Fe²⁺ in haemoglobin is unreactive, this avoids potentially deleterious catalysis of free radical-mediated lipid peroxidation of cell membranes, which occurs in rice cells during post-thaw recovery [[26]]. In general, haemoglobins derived from animal red blood cells (e.g. Erythrogen™ supplied by Biorelease Corporation, Salem, USA; Table 5) that are available commercially for incorporation into cell culture media are treated chemically with pyridoxal-5-phosphate to lower their oxygen binding to acceptable physiological levels.

Table V. Properties of Erythrogen™

Total haemoglobin content (g 100 ml^−1)	10.3
P50 (Torr at 37°C)	38.0
Methaemoglobin content (%)	< 4
pH	7.42
Stable ca. 1 year at 5–10°C

In plant systems, supplementation of the culture medium with Erythrogen™ at a concentration of 1:50–1:500 (v:v), had beneficial effects on the post-thaw growth following cryopreservation for 30 days of suspension cultured cells of the Indica rice cv. Pusa Basmati 1. The mean absorbance, following TTC reduction, of the rice cells at 8 days after thawing was increased by up to 60% (P < 0.05) over the control. Erythrogen™ at 1:50–1:100 (v:v) promoted sustained mitotic division, with cell biomass at 24 days following thawing being increased up to 22% (P < 0.05) above the control. An interesting fact was that Pusa Basmati 1 suspensions re-initiated from cryopreserved cells recovered in the presence of Erythrogen™ exhibited significantly faster growth rates over a 20 day culture period compared with cell suspensions that had been recovered without Erythrogen™. This stimulation of growth is important, since it reduces the time required for the re-establishment of suspension cultures of cryopreserved cell lines, with concomitant maintenance of cell totipotency. The maintenance of totipotency is particularly relevant to studies involving the genetic manipulation of cells per se, or following conversion to protoplasts. Importantly, the yields of viable protoplasts from cryopreserved cells recovered in the presence of Erythrogen™ were comparable to those of unfrozen cultures. Whilst in depth studies are required with cells of plants other than rice, the present results imply that the commercial preparation Erythrogen™ can be incorporated routinely into culture media to increase plant cell recovery and growth during post-thaw handling procedures.

Further investigations are also essential to determine the precise mechanism(s) by which haemoglobins, such as Erythrogen™, facilitate the post-thaw survival and growth of plant cells. A focus of such work should be to study the effects of Erythrogen™ on respiratory gas dynamics, since previous results demonstrated that respiratory imbalances occur during the initial post-thaw phase [24-25]. In this respect, mitochondria isolated from thawed rice cells exhibited the same degree of coupling of mitochondrial electron transport with ATP synthesis as unfrozen cells [[24]]. Erythrogen™ may enhance mitochondrial oxygen consumption, leading to increases in cellular ATP and related metabolites. In previous work with animal cells, Shi et al. [[87]] demonstrated that Erythrogen™ not only enhanced cell division, but also stimulated increased production of recombinant protein. Erythrogen™ is believed to “trap” oxygen from air-medium interfaces, facilitating delivery of the gas to cultured cells. In addition to alterations in oxygen supply per se, it is possible that the growth enhancement produced by supplementing medium with Erythrogen™ may be part-driven by subtle changes in iron availability to cells, mild buffering of the pH of the medium, or the uptake of amino acids from hydrolysed haemoglobin. Future studies should clarify the context in which these parameters enhance cell growth. Additionally, as demonstrated for PFC, it will be essential to assess whether any synergistic effects of Pluronic® F-68 with Erythrogen™ in the recovery medium for cells after thawing, enhances mitotic division of cultured protoplasts of Petunia hybrida [[88]].

CONCLUDING REMARKS

Experiments using cryopreserved rice cells, as a model totipotent eukaryotic system, have focused on the optimisation of the post-thaw conditions for the successful recovery of suspension cells from long-term storage in liquid nitrogen. However, these investigations did not address the basic requirement of providing thawed cells with an adequate supply of oxygen, particularly in the early and crucial stages of post-thaw recovery. It is at this critical stage that oxidative stress conditions arise leading to the production of toxic oxygen radicals, which are well known to cause lipid peroxidation of the cells. This, in turn, can lead to rancidification of adjacent cells which, in some cases, may result in unsuccessful recovery of cells from cryopreservation, through insufficient viable cells being available to enter sustained mitotic division. The novel technologies described in this review underpin this earlier work and should now be extended to cells and tissues of species that respond poorly to conventional post-thaw recovery strategies. Indeed, it is possible that cells of plants which, to date, have been considered non-recoverable from cryopreservation can now be recovered successfully using these novel approaches.

In conclusion, whilst the data are still somewhat limited, there is a considerable body of evidence that non-ionic surfactants, PFCs and chemically-modified haemoglobin stimulate significantly the post-thaw viability and biomass production of cultured plant cells. The general applicability of the use of these compounds, either alone or in combination, will become clear once they are extended to a range of plant cells, tissues and organs. The incorporation of these simple and, with the exception of PFCs, relatively inexpensive compounds into plant systems, results from initial unequivocal demonstration of their applications to animal cell systems. Such investigations emphasise the need, even in the development of cryopreservation protocols, for plant workers to be aware of, and to benefit from, the most recent advances reported in the animal-based literature.