The objective of this thesis was to investigate and expand the use of testis tissue xenografting as means of maintaining the developmental potential of donor testis tissue. The objective of the first study was to investigate the effect of donor age on spermatogenesis in canine testis tissue after xenografting into immunodeficient recipient mice. Fragments of testis tissue from 12 dogs of different ages were xenografted under the back skin of mice. Donors were categorized based on testis developmental status at the time of grafting into: less than four months (immature), four to six months (young), and greater than six months of age (adult). The grafts were evaluated at four, six or eight months post-grafting. At four months post-grafting, immature and young groups had higher graft recovery rates (92 ± 5.8 and 88 ± 4.4% versus 69 ± 3.5%; P = 0.001 and P = 0.001), graft weights (34 ± 8.1 and 32 ± 11.0 mg versus 7 ± 2.6 mg; P = 0.001 and P = 0.02), vesicular gland indices (1.1 ± 0.20 and 0.6 ± 0.18% versus 0.1 ± 0.03%; P < 0.0001 and P = 0.02), seminiferous tubule numbers (517 ± 114.8 and 364 ± 161.0 versus 10 ± 5.1; P < 0.0001 and P = 0.03), and larger seminiferous tubular diameters (140 ± 17.8 and 130 ± 3.4 µm versus 55 ± 21.9 µm; P = 0.003 and P = 0.001), compared to adult donor xenografts. Xenografts from immature donors maintained the growth and development for eight months, as exhibited by greater graft weights (17 ± 4.6 mg, P = 0.002), seminiferous tubule numbers (547 ± 210.3, P < 0.01) and tubular diameters (93 ± 15.9 µm, P < 0.0001), and induced greater vesicular gland indices (1.5 ± 0.46%, P = 0.0005), compared to adult donor xenografts. The growth and development of testis tissue xenografts from immature and young donors were not different after eight months (P > 0.05). Young donor xenografts had greater seminiferous tubule number and diameter compared to adult donor xenografts (P = 0.009 and P = 0.004, respectively) at eight months post-grafting. Elongated spermatids were the most advanced germ cell type present at four and eight months post-grafting in the testis grafts of immature and young age groups.<p> The objective of the second study was to evaluate three different strategies to preserve/cryopreserve immature porcine testis tissue. Immature porcine testes were cooled at 4 °C for 24, 48 or 72 hours, and testis tissue fragments were cryopreserved using programmed slow freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, or vitrified using DMSO or glycerol at 5, 15 or 30 min exposure time. In vitro cell viability was determined by trypan blue exclusion, and in vivo developmental potential was evaluated by xenografting into immunodeficient mice. Compared to fresh tissue, short-term cooling of porcine testis tissue resulted in similar in vitro cell survival rates (93 ± 2.2% for fresh versus 95 ± 0.3, 93 ± 1.7 and 87 ± 4.3% after 24, 48 and 72 hours at 4 °C, respectively; P = 0.74) and in vivo development, with generation of elongated spermatids and sperm after four months of grafting. Cryopreservation of testis tissue with programmed slow freezing using glycerol and vitrification with DMSO (5 min equilibration) or glycerol (5 or 15 min equilibration) did not compromise the developmental competence of xenografts when compared to fresh tissue (control), characterized by the formation of elongated spermatids and sperm.<p> These findings suggest that canine testis tissue from immature donors and cooling of immature porcine testis tissue to refrigerator temperature for up to 72 hours or cryopreservation with slow controlled freezing or vitrification could be suitable methods to restore male fertility following xenografting.

The objective of this thesis was to investigate and expand the use of testis tissue xenografting as means of maintaining the developmental potential of donor testis tissue. The objective of the first study was to investigate the effect of donor age on spermatogenesis in canine testis tissue after xenografting into immunodeficient recipient mice. Fragments of testis tissue from 12 dogs of different ages were xenografted under the back skin of mice. Donors were categorized based on testis developmental status at the time of grafting into: less than four months (immature), four to six months (young), and greater than six months of age (adult). The grafts were evaluated at four, six or eight months post-grafting. At four months post-grafting, immature and young groups had higher graft recovery rates (92 ± 5.8 and 88 ± 4.4% versus 69 ± 3.5%; P = 0.001 and P = 0.001), graft weights (34 ± 8.1 and 32 ± 11.0 mg versus 7 ± 2.6 mg; P = 0.001 and P = 0.02), vesicular gland indices (1.1 ± 0.20 and 0.6 ± 0.18% versus 0.1 ± 0.03%; P < 0.0001 and P = 0.02), seminiferous tubule numbers (517 ± 114.8 and 364 ± 161.0 versus 10 ± 5.1; P < 0.0001 and P = 0.03), and larger seminiferous tubular diameters (140 ± 17.8 and 130 ± 3.4 µm versus 55 ± 21.9 µm; P = 0.003 and P = 0.001), compared to adult donor xenografts. Xenografts from immature donors maintained the growth and development for eight months, as exhibited by greater graft weights (17 ± 4.6 mg, P = 0.002), seminiferous tubule numbers (547 ± 210.3, P < 0.01) and tubular diameters (93 ± 15.9 µm, P < 0.0001), and induced greater vesicular gland indices (1.5 ± 0.46%, P = 0.0005), compared to adult donor xenografts. The growth and development of testis tissue xenografts from immature and young donors were not different after eight months (P > 0.05). Young donor xenografts had greater seminiferous tubule number and diameter compared to adult donor xenografts (P = 0.009 and P = 0.004, respectively) at eight months post-grafting. Elongated spermatids were the most advanced germ cell type present at four and eight months post-grafting in the testis grafts of immature and young age groups.<p> The objective of the second study was to evaluate three different strategies to preserve/cryopreserve immature porcine testis tissue. Immature porcine testes were cooled at 4 °C for 24, 48 or 72 hours, and testis tissue fragments were cryopreserved using programmed slow freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, or vitrified using DMSO or glycerol at 5, 15 or 30 min exposure time. In vitro cell viability was determined by trypan blue exclusion, and in vivo developmental potential was evaluated by xenografting into immunodeficient mice. Compared to fresh tissue, short-term cooling of porcine testis tissue resulted in similar in vitro cell survival rates (93 ± 2.2% for fresh versus 95 ± 0.3, 93 ± 1.7 and 87 ± 4.3% after 24, 48 and 72 hours at 4 °C, respectively; P = 0.74) and in vivo development, with generation of elongated spermatids and sperm after four months of grafting. Cryopreservation of testis tissue with programmed slow freezing using glycerol and vitrification with DMSO (5 min equilibration) or glycerol (5 or 15 min equilibration) did not compromise the developmental competence of xenografts when compared to fresh tissue (control), characterized by the formation of elongated spermatids and sperm.<p> These findings suggest that canine testis tissue from immature donors and cooling of immature porcine testis tissue to refrigerator temperature for up to 72 hours or cryopreservation with slow controlled freezing or vitrification could be suitable methods to restore male fertility following xenografting.

The long-term preservation of fungi has been carried out using several different methods, depending on resources and laboratory. Isolates have been deposited in culture collections for preservation and storage for periods of many years. Despite the widespread use of preservation regimes, little attention has been paid to the poststorage stability of the physiological and genetic characters of strains. The loss of viability of a biological control agent or the failure of an isolate to produce a secondary metabolite pivotal in the production of drugs or food could result in substantial economic loss for the manufacturing organisation. In this investigation the effects of five preservation protocols on fungal characters were assessed: continual sub-culture, lyophilisation, storage in water, storage at -20°C and cryopreservation in liquid nitrogen The physiology and genetic stability of three species of economically important fungi (Metarhizium anisopliae, Fusarium oxysporum and Serpula lacrymans) were examined by analysis of culture characteristics, secondary metabolite profiling, extracellular enzyme tests and PCR fingerprinting over a two-year testing period. It was found that preservation regime can influence the resultant characters of the test fungi. Radial growth rate and conidial production was changed from the original isolates after preservation and storage. Secondary metabolite profiles from all of the test fungi were susceptible to change by the preservation protocols assessed. Production of some metabolites was lost, whereas others remained stable after preservation and storage. Extracellular enzyme production was also affected in a similar way. For example, some replicates of an isolate of Metarhizium anisopliae lost ~-galactosidase activity after one year of preservation. Genetic stability was also compromised is some isolates. Polymorphisms were detected after PCR fingerprinting with a micro-satellite primer in replicates of two isolates of Metarhizium anisopliae that had been stored for one and two years by cryopreservation and lyophilisation and in two replicates of an isolate of Fusarium oxysporum maintained by continual sub-culture for sixteen weeks. The results indicate that response to preservation and storage is species- and strain-specific. Therefore, there is a need to develop new and existing preservation regimes with emphasis on strain-specific criteria. Scientists should preserve their important isolates by more than one preservation method to protect organisms from the stresses encountered during preservation and storage.

Sugarcane has become an increasingly important crop in recent years, with South Africa featuring as one of the prominent producers. This has led to a significant growth in the South African sugarcane industry, translating into an increased demand for planting material. Although this demand is now satisfied by recent biotechnological advancements such as protocols for somatic embryogenesis to increase the production of planting material, such techniques are limited as a result of the progressive loss of the embryogenic potential of calli over time. In order to facilitate management of this material, it is desirable to develop a protocol for the long-term storage of the germplasm. This study reports on investigations of the different parameters that influenced the cryoprocess in attempts to develop a protocol for the successful cryopreservation of sugarcane somatic embryos of the 88H0019 variety. Experiments were carried out to determine in vitro culture conditions for successful induction of somatic embryos via both the direct and indirect routes of micropropagation. A suitable regeneration medium for plantlet establishment pre- and post-cooling was established (Chapter 2). Investigations were also carried out to ascertain the responses of somatic embryos to both rapid and slow dehydration techniques (Chapter 3). Finally, several cooling techniques (both slow and rapid), were applied, on partially dehydrated somatic embryos, either without, or after cryoprotection, in an attempt to achieve survival after cryopreservation of the somatic embryos (Chapter 4). Both directly- and indirectly-derived somatic embryos were converted, most successfully, on full strength Murashige and Skoog medium without addition of plant growth regulators. The initial mean water contents of directly- and indirectly-derived somatic embryos were not significantly different from each other (8.38±0.19 g g-1 and 8.45±0.33 g g-1 [dry mass basis], respectively). The percentage conversion at these water contents was also not significantly different; 97% for directly- and 98% for indirectlyinduced embryos. Slow dehydration by culture on a series of media with increasing concentrations of sucrose (from 0.2 M to 1.2 M) for a period of 48 h each was the most effective technique, with water content being reduced to 0.94±0.03 g g-1 and 0.95±0.02 g g-1 after dehydration on media containing 1.0 M sucrose, while maintaining between 98% and 100% conversion, respectively. Of the various cryoprotectants tested, proline and casamino acid had the least adverse effects on the somatic embryos. The encapsulation-vitrification cooling technique was the most efficient of all techniques employed. The best conditions involved encapsulation of embryo clumps in a solution of MS medium with 3% (w/v) Na-alginate and loading solution containing 2 M glycerol plus 0.4 M sucrose, followed by infiltration and dehydration at 0°C for various time intervals (0, 5, 10, 15, 20, 25, 30 min) with 1 ml PVS2 solution and thereafter, rapid immersion in liquid nitrogen. Under such conditions, 30% of the cryopreserved somatic embryos retained viability, going on to form callus from which shoots and roots were produced. Although somatic embryos of sugarcane of the local variety 88H0019 have proved to be recalcitrant to cryopreservation, the results obtained with explants that had been processed by encapsulation-vitrification suggest that this approach may be worth pursuing and refining. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.

No description available.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The Australian almond improvement program was initiated in 1997 to develop improved cultivars that are adapted to local conditions and consumer demands. The program combines molecular techniques along with the traditional approach of controlled hybridisation with mass selection. This research project was carried out to assist the Australian almond improvement program in the areas of micropropagation, cryopreservation, and rapid identification of self-incompatibility genotypes of almond. Micropropagation was accomplished successfully for two commercially important almond cultivars ('Nonpareil' 15-1 and 'Ne Plus Ultra') and an almond/peach hybrid rootstock by culturing shoot tips, about 0.7 cm long with 3 - 5 leaves, on appropriate shoot multiplication media. For 'Nonpareil' 15-1, AP medium with 0.049 uM IBA, 3 uM BAP, 0.058 M sucrose, and 0.7% agar at pH 5.7 was effective. MS medium with 0.049 uM IBA, 5 uM BAP, 0.088 M sucrose, and 0.7% agar at pH 5.7 was suitable for 'Ne Plus Ultra'. For the almond/peach hybrid rootstock, MS medium supplemented with 10 uM BAP, 0.088 M sucrose, and 0.7% agar provided the best shoot proliferation. Shoots of the rootstock, about two cm long, readily produced roots after one week in the dark and two weeks in the light on half strength MS medium supplemented with 2.4 uM IBA, 0.088 M sucrose and 0.7% agar at pH 5.7, with 88.0% rooting efficiency. The two almond cultivars did not readily produce roots, but, at about 1.5 cm long, were micrografted successfully onto the rootstock. These micrografted plantlets were acclimatised and transferred to potting mix with 92% survival. Shoot tips of the two almond cultivars and the almond/peach hybrid rootstock were cryopreserved successfully using a one-step vitrification technique. Three-week-old in vitro cultures were cold-hardened at 4°C on multiplication media (Murashige and Skoog for 'Ne Plus Ultra' and the hybrid rootstock; Almehdi and Parfitt for 'Nonpareil' 15-1) for three weeks. Shoot tips, 2 - 2.5 mm long, were excised and precultured for one day at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond cultivars and 60 min for the almond/peach hybrid rootstock, and then stored under liquid nitrogen (LN) for up to 24 months. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄N0₃ or (NH₄)₂S0₄. Shoot regeneration was usually observed within 2 - 3 weeks. Survival of shoots after thawing varied from 56-80% for 'Ne Plus Ultra', 35-53% for 'Nonpareil' 15-1, and 62 - 82% for the almond/peach hybrid rootstock. Non-vitrified shoots that were stored on basal medium at 3.5-5°C showed good survival up to six months, but thereafter survival decreased rapidly. Cryopreservation has considerable potential for long-term storage of almond germplasm, but future research should be aimed at improving the regeneration of 'Nonpareil' 15-1, the most important commercial cultivar grown in Australia. The genetic stability of almond DNA to both in vitro culture and the cryopreservation process was evaluated by comparing the fingerprints of the DNA from the original orchard trees, from the in vitro cultures before and after cryopreservation for up to 24 months, and from plants regenerated from in vitro cultures. The fingerprints were prepared by initially digesting the DNA with two isoschizomer pairs of restriction enzymes, one of each pair being 'methylation sensitive' and the other 'methylation insensitive', followed by amplification of the digested products using randomly amplified polymorphic DNA (RAPD) with six different 10-mer primers. Changes in methylation were found between the original orchard trees and in vitro cultures, and there was also the possibility that some structural changes may have occurred. However, no methylation or structural changes could be attributed to the cryopreservation procedure. Plants regenerated from the in vitro cultures before and after cryopreservation should be monitored carefully in the future for changes in morphology compared to the original trees. Partial genomic and cDNA sequences of the self-incompatibility alleles S1, S2, S7, S8, S9, S10, S23, and Sf were obtained from Prunus dulcis cvs 'Anxaneta' (S2S9), 'Cristomorto' (S1S2), 'Ferragnes' (S1S3), 'Gabaix' (S5S10), 'Ne Plus ultra' (S/S7), 'Nonpareil' 15-1 (S7S8), 'Primorskiy'(S5S9), 'Ramilette' (S6S23), and IRTA Selection 12-2 (SfSf). Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from 'Ferragnes', and the S7 and S8 alleles from 'Nonpareil' 15-1 matched those reported in the literature for the alleles Sb, Sc, and Sd respectively. The sequences of the S1, S2, S7, S8, S9, S10, S23, and Sf alleles found in genomic DNA contained introns of 562, 253, 1,530, 2,208, 1,343, 710, 494, and 662 bp respectively, and partial exons of 510, 537, 489, 498, 486, 495, 489, and 543 bp respectively. In addition, one allele of the Australian cultivars, 'Johnston's Prolific' and 'Pierce', was identified and found to have the same sequence as S23 in 'Ramilette', suggesting that this cultivar may have been an early introduction to Australia from Spain. The exon/intron splice junction sites of all alleles followed the GT / AG consensus sequence rule, and the sequences were found to be highly conserved. Both the length and the sequence of each intron was unique, and a technique of identifying the S-alleles of almond was developed based on primers that targetted the intron sequences. The use of these primers has increased the speed, precision, and efficiency with which the incompatilibity genotypes of almond cultivars can be detected, compared to other published techniques. The primers confirmed the S-allele specificities for 26 out of 30 cultivars for which published information is available, and are currently in use in the Australian almond improvement program to identify incompatibility groups in the breeding progeny. Future work should be directed towards obtaining the sequences of the introns for the remaining known S-alleles, S3 to S6, and S11 to S22. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1051244 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, 2002

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The Australian almond improvement program was initiated in 1997 to develop improved cultivars that are adapted to local conditions and consumer demands. The program combines molecular techniques along with the traditional approach of controlled hybridisation with mass selection. This research project was carried out to assist the Australian almond improvement program in the areas of micropropagation, cryopreservation, and rapid identification of self-incompatibility genotypes of almond. Micropropagation was accomplished successfully for two commercially important almond cultivars ('Nonpareil' 15-1 and 'Ne Plus Ultra') and an almond/peach hybrid rootstock by culturing shoot tips, about 0.7 cm long with 3 - 5 leaves, on appropriate shoot multiplication media. For 'Nonpareil' 15-1, AP medium with 0.049 uM IBA, 3 uM BAP, 0.058 M sucrose, and 0.7% agar at pH 5.7 was effective. MS medium with 0.049 uM IBA, 5 uM BAP, 0.088 M sucrose, and 0.7% agar at pH 5.7 was suitable for 'Ne Plus Ultra'. For the almond/peach hybrid rootstock, MS medium supplemented with 10 uM BAP, 0.088 M sucrose, and 0.7% agar provided the best shoot proliferation. Shoots of the rootstock, about two cm long, readily produced roots after one week in the dark and two weeks in the light on half strength MS medium supplemented with 2.4 uM IBA, 0.088 M sucrose and 0.7% agar at pH 5.7, with 88.0% rooting efficiency. The two almond cultivars did not readily produce roots, but, at about 1.5 cm long, were micrografted successfully onto the rootstock. These micrografted plantlets were acclimatised and transferred to potting mix with 92% survival. Shoot tips of the two almond cultivars and the almond/peach hybrid rootstock were cryopreserved successfully using a one-step vitrification technique. Three-week-old in vitro cultures were cold-hardened at 4°C on multiplication media (Murashige and Skoog for 'Ne Plus Ultra' and the hybrid rootstock; Almehdi and Parfitt for 'Nonpareil' 15-1) for three weeks. Shoot tips, 2 - 2.5 mm long, were excised and precultured for one day at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond cultivars and 60 min for the almond/peach hybrid rootstock, and then stored under liquid nitrogen (LN) for up to 24 months. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄N0₃ or (NH₄)₂S0₄. Shoot regeneration was usually observed within 2 - 3 weeks. Survival of shoots after thawing varied from 56-80% for 'Ne Plus Ultra', 35-53% for 'Nonpareil' 15-1, and 62 - 82% for the almond/peach hybrid rootstock. Non-vitrified shoots that were stored on basal medium at 3.5-5°C showed good survival up to six months, but thereafter survival decreased rapidly. Cryopreservation has considerable potential for long-term storage of almond germplasm, but future research should be aimed at improving the regeneration of 'Nonpareil' 15-1, the most important commercial cultivar grown in Australia. The genetic stability of almond DNA to both in vitro culture and the cryopreservation process was evaluated by comparing the fingerprints of the DNA from the original orchard trees, from the in vitro cultures before and after cryopreservation for up to 24 months, and from plants regenerated from in vitro cultures. The fingerprints were prepared by initially digesting the DNA with two isoschizomer pairs of restriction enzymes, one of each pair being 'methylation sensitive' and the other 'methylation insensitive', followed by amplification of the digested products using randomly amplified polymorphic DNA (RAPD) with six different 10-mer primers. Changes in methylation were found between the original orchard trees and in vitro cultures, and there was also the possibility that some structural changes may have occurred. However, no methylation or structural changes could be attributed to the cryopreservation procedure. Plants regenerated from the in vitro cultures before and after cryopreservation should be monitored carefully in the future for changes in morphology compared to the original trees. Partial genomic and cDNA sequences of the self-incompatibility alleles S1, S2, S7, S8, S9, S10, S23, and Sf were obtained from Prunus dulcis cvs 'Anxaneta' (S2S9), 'Cristomorto' (S1S2), 'Ferragnes' (S1S3), 'Gabaix' (S5S10), 'Ne Plus ultra' (S/S7), 'Nonpareil' 15-1 (S7S8), 'Primorskiy'(S5S9), 'Ramilette' (S6S23), and IRTA Selection 12-2 (SfSf). Total DNA was extracted from leaves, and cDNA was prepared from total RNA extracted from styles. The partial cDNA sequences of the S1 allele from 'Ferragnes', and the S7 and S8 alleles from 'Nonpareil' 15-1 matched those reported in the literature for the alleles Sb, Sc, and Sd respectively. The sequences of the S1, S2, S7, S8, S9, S10, S23, and Sf alleles found in genomic DNA contained introns of 562, 253, 1,530, 2,208, 1,343, 710, 494, and 662 bp respectively, and partial exons of 510, 537, 489, 498, 486, 495, 489, and 543 bp respectively. In addition, one allele of the Australian cultivars, 'Johnston's Prolific' and 'Pierce', was identified and found to have the same sequence as S23 in 'Ramilette', suggesting that this cultivar may have been an early introduction to Australia from Spain. The exon/intron splice junction sites of all alleles followed the GT / AG consensus sequence rule, and the sequences were found to be highly conserved. Both the length and the sequence of each intron was unique, and a technique of identifying the S-alleles of almond was developed based on primers that targetted the intron sequences. The use of these primers has increased the speed, precision, and efficiency with which the incompatilibity genotypes of almond cultivars can be detected, compared to other published techniques. The primers confirmed the S-allele specificities for 26 out of 30 cultivars for which published information is available, and are currently in use in the Australian almond improvement program to identify incompatibility groups in the breeding progeny. Future work should be directed towards obtaining the sequences of the introns for the remaining known S-alleles, S3 to S6, and S11 to S22. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1051244 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, 2002

No description available.

Thesis (Ph. D.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Sambanis, Athanassios; Committee Member: Long, Jr., Robert C.; Committee Member: Ludovice, Peter J.; Committee Member: Prausnitz, Mark R.; Committee Member: Song, Ying C.

Ice recrystallization during thawing after cryopreservation results in extensive cellular damage that ultimately leads to cell death and decreased cell viabilities. This is a significant problem particularly with cryopreserved cells utilized in various regenerative medicine therapies. Given the success of these therapies to treat spinal cord injury, cartilage lesions, and cardiacdisease, the development of new and improved cryprotectants that minimize cell damageduring freeze-thawing and improve cell viability post-cryopreservation are urgently required. The current cryopreservative dimethyl sulfoxide, DMSO, is associated with cytotoxicity in clinical settings and is not an optimal cryopreservative. Our laboratory is interested in synthesizing small molecules that possess the property of ice recrystallization inhibition (IRI) activity that can be utilized as cryopreservatives without the cytotoxic effects associated with DMSO. This thesis focuses on the development of small molecule ice recrystallization inhibitors and elucidating the structural features of disaccharides and surfactants that are responsible for potent IRI activity. The first part of this study examines simple disaccharide derivatives mimicking those found in the native AFGP to determine whether disaccharide structure influences IRI activity. Towards this end, the (1,6)-linked AFGP disaccharide analogue was synthesized, assessed for IRI activity using a splat-cooling assay, and compared to the native (1,3)- and (1,4)-linked AFGP disaccharide analogues. The change in linkage was found to have a profound affect on IRI activity. The second part of the study focuses on surfactants and gelators as ice recrystallization inhibitors. Our laboratory has demonstrated that carbohydrate-based hydrogelators can be potent inhibitors of ice recrystallization. While our studies have indicated that a delicate balance between hydrophobic and hydrophilic interactions is crucial for ice recrystallization inhibition (IRI) activity, the essential structural features necessary for potent IRI activity remain unknown. To address this issue, structurally diverse amino acid-based surfactants/gelators, anti-ice nucleating agents, and glycoconjugates were synthesized and assessed for IRI activity. The results indicate that long alkyl chains and increased hydrophobicity are important for potent IRI activity and iii that the position of these alkyl chains is essential. Also, the counterion of these compounds affects the IRI activity and is related to the counterion degree of hydration. These compounds were assessed for their ability to cryopreserve human liver cells (Hep G2) and human bone marrow cells (Tf-1α) in cell-based assays. Additionally, the best IRI assay solution was determined, which involved studying how the salts of the phosphate buffered saline (PBS) solution modulated IRI activity. Finally, small molecule ice recrystallization inhibitors were assessed for their ability to protect the viral vectors vaccinia virus, vesicular stomatitis virus, and herpes simplex-1 virus at various storage conditions. This will aid in developing improved preservation protocols for vaccines and viruses utilized in cancer therapy (oncolytic viruses).