**1. Introduction**

Cryopreservation is an established practice of freezing and storing valuable biological materials in liquid nitrogen for long periods of time for use in research, medicine, environ‐ mental studies, and technology development. These materials include parasites, vector tissues,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and organs, a wide range of human stem cells, plants, microorganisms, etc. Research on these materials assists in understanding how ecosystems function, how disease transmission takes place, how human bodies function, and why some vectors of the same species are efficient at disease transmission whereas others are not able to transmit. Recognizing the importance of collections of biological materials to research and development and acknowledging the high cost of field sample collection in terms of financial resources and time, the management of the Kenya Trypanosomiasis Research Institute (KETRI) has put in place an institutional policy of continuous collection of trypanosome parasites for cryopreservation. This took advantage of all field visits undertaken by various scientific research teams to different foci in Kenya, a country that is endemic for both human and animal trypanosomiasis. This resulted in the establishment of the KETRI Trypanosome Bank which currently has over 2000 isolates [1] from various hosts (tsetse flies, human, domestic and wild animals). Some of the recent collections include vectors of trypanosomiasis, the tsetse flies. Updating of the cryo‐bank with fresh trypanosome isolates is a continuous process.

Trypanosomes are extracellular protozoan parasites which cause debilitating disease in humans and animals. In humans, the disease is referred to as human African trypanosomiasis (HAT) or sleeping sickness, caused by two trypanosome species, *Trypanosoma brucei gam‐ biense*, responsible for the chronic form of HAT in West and Central Africa, and *T. b. rhode‐ siense*, which causes acute disease in eastern and southern Africa. The parasites are transmitted by tsetse flies (*Glossina* spp.). In animals, the disease is referred to as African animal trypano‐ somiasis (AAT; nagana in cattle, sheep, and goats; surra in camels) and is caused by various trypanosome species, the major ones being *T. vivax*, *T. congolense* and *T. evansi* [2]. Whereas majority of the trypanosome species which cause AAT are transmitted by tsetse fly vectors, *T. evansi*, is transmitted mechanically by biting flies such as *Tabanus* spp.; *T. vivax* has been reported to be transmitted by both tsetse flies and biting flies [3].

HAT is classified in the category of the most neglected tropical diseases. Current diagnostic tools have inadequate sensitivity and specificity, thus complicating disease diagnosis and staging. The drugs available for treatment are highly toxic and not very effective; patients die if untreated [4, 5]. In 2005, an annual prevalence of 50–70,000 HAT cases/year was reported, with incidence rates of 15–17,000 cases/year [6]. Although recent data from the World Health Organization (WHO) shows that the number of reported cases of HAT declined to less than 10,000 in 2009 leading to speculation that the disease could be eliminated [7, 8], there is great need to maintain vigilance through surveillance and research. This is informed by the fact that HAT was effectively controlled in the 1960s in many endemic countries; however, the disease re‐surged due to breakdown in surveillance and control activities (**Figure 1**). WHO [9] has developed a roadmap for elimination of HAT by the year 2020, which involves development of new and better diagnostics and drugs [5, 8]. Cryo‐banks such as the KETRI Trypanosome Bank will therefore be important in contributing to this strategy in order to ensure that epidemics do not occur in future; and that dormant foci will be prioritized for elimination. One of the issues for which answers are sought is what happens in some traditional HAT foci when the disease is not reported in humans. Some of the new technological advances that are providing more insights include genetic analysis of both parasite and vector genomes and identification of specific proteins as targets for development of vaccines, new and sensitive diagnostic tests.

**Figure 1.** Sleeping sickness as a reemerging disease.

and organs, a wide range of human stem cells, plants, microorganisms, etc. Research on these materials assists in understanding how ecosystems function, how disease transmission takes place, how human bodies function, and why some vectors of the same species are efficient at disease transmission whereas others are not able to transmit. Recognizing the importance of collections of biological materials to research and development and acknowledging the high cost of field sample collection in terms of financial resources and time, the management of the Kenya Trypanosomiasis Research Institute (KETRI) has put in place an institutional policy of continuous collection of trypanosome parasites for cryopreservation. This took advantage of all field visits undertaken by various scientific research teams to different foci in Kenya, a country that is endemic for both human and animal trypanosomiasis. This resulted in the establishment of the KETRI Trypanosome Bank which currently has over 2000 isolates [1] from various hosts (tsetse flies, human, domestic and wild animals). Some of the recent collections include vectors of trypanosomiasis, the tsetse flies. Updating of the cryo‐bank with fresh

Trypanosomes are extracellular protozoan parasites which cause debilitating disease in humans and animals. In humans, the disease is referred to as human African trypanosomiasis (HAT) or sleeping sickness, caused by two trypanosome species, *Trypanosoma brucei gam‐ biense*, responsible for the chronic form of HAT in West and Central Africa, and *T. b. rhode‐ siense*, which causes acute disease in eastern and southern Africa. The parasites are transmitted by tsetse flies (*Glossina* spp.). In animals, the disease is referred to as African animal trypano‐ somiasis (AAT; nagana in cattle, sheep, and goats; surra in camels) and is caused by various trypanosome species, the major ones being *T. vivax*, *T. congolense* and *T. evansi* [2]. Whereas majority of the trypanosome species which cause AAT are transmitted by tsetse fly vectors, *T. evansi*, is transmitted mechanically by biting flies such as *Tabanus* spp.; *T. vivax* has been

HAT is classified in the category of the most neglected tropical diseases. Current diagnostic tools have inadequate sensitivity and specificity, thus complicating disease diagnosis and staging. The drugs available for treatment are highly toxic and not very effective; patients die if untreated [4, 5]. In 2005, an annual prevalence of 50–70,000 HAT cases/year was reported, with incidence rates of 15–17,000 cases/year [6]. Although recent data from the World Health Organization (WHO) shows that the number of reported cases of HAT declined to less than 10,000 in 2009 leading to speculation that the disease could be eliminated [7, 8], there is great need to maintain vigilance through surveillance and research. This is informed by the fact that HAT was effectively controlled in the 1960s in many endemic countries; however, the disease re‐surged due to breakdown in surveillance and control activities (**Figure 1**). WHO [9] has developed a roadmap for elimination of HAT by the year 2020, which involves development of new and better diagnostics and drugs [5, 8]. Cryo‐banks such as the KETRI Trypanosome Bank will therefore be important in contributing to this strategy in order to ensure that epidemics do not occur in future; and that dormant foci will be prioritized for elimination. One of the issues for which answers are sought is what happens in some traditional HAT foci when the disease is not reported in humans. Some of the new technological advances that are providing more insights include genetic analysis of both parasite and vector genomes and

trypanosome isolates is a continuous process.

4 Cryopreservation in Eukaryotes

reported to be transmitted by both tsetse flies and biting flies [3].

Isolation and cryopreservation of new trypanosome strains from patients in different HAT foci ensures availability of these stabilates for use in parasitological, biochemical, molecular, serological and pharmacological investigations many years after their isolation from the host. Brun *et al* [2] observed that one of the major obstacles in the elucidation of the factors responsible for relapses after melarsoprol treatment was the lack of recent *T. b. gambiense* isolates from patients from various endemic areas where the problem has been reported. The WHO steering committee on human African trypanosomiasis treatment has therefore recommended that collection of stabilates be a continuous activity in order to monitor the occurrence and spatial distribution of treatment failure [10] and refractoriness of tsetse to infection. Since its inception, KETRI and now KALRO‐Biotechnology Research Institute developed an institutional policy of encouraging collection of stabilates by scientists and clinicians, for cryopreservation. In this chapter, we describe the procedures of isolation and cryopreservation of trypanosome stabilates for research and development in resource constrained settings.
