**9. Terminologies commonly associated with cryopreservation (from: Trypanosomiasis a veterinary perspective Lorne E. Stephen pages 440–441)**

Terminologies commonly used with cryopreservation technique

**1.** Trypanosome species

Assemblages of organisms that can be distinguished from other species by one or more stable discontinuous morphological characters, e.g., *T. congolense*, *T. vivax*, *T. brucei* are different species.

**2.** Trypanosome subspecies

Assemblages of organisms within a species that cannot be separated from each other by morpho‐ logical characters but only by other stable characters, e.g., *T. b. rhodesiense*, *T. b. gambiense* are subspecies of *T. brucei*.

#### **3.** Clone

This is a population that has been developed from a single trypanosome.

**4.** Line

A laboratory derivative of a stock maintained in different physical conditions, e.g., a species of *T. congolense* maintained in mice only is a line of that nature, while when maintained in cattle only is another line of that nature, etc.

**5.** Population

The group of trypanosomes present at a given time in a given host or it may consist of a mixture of several species and subspecies.

**6.** Primary isolate

This is a stabilate made from a naturally infected host or viable organisms present in a culture or experimental animals following the introduction of the sample from a naturally infected host.

**7.** Sample

That part of trypanosome population collected on a unique occasion.

**8.** Stabilate

A cryopreserved sample of viable trypanosomes.

**9.** Stock

A population derived by serial passage *in vivo* or *in vitro* from a primary isolate.

**10.** Metacyclics

These are the mammalian infective forms of trypanosomes injected by the tsetse fly. Metacyclic forms are found at the end of transmission cycle.

**11.** Procyclics

These are the midgut forms of trypanosomes which are found in cultures.

#### **10. Conclusion**

Communicable and noncommunicable diseases, including the neglected tropical diseases, cause chronic life‐long disability, hinder economic development, and impair childhood development in resource poor settings in Africa where the diseases are endemic [27, 28]. Control of these diseases could be an efficient way to fight poverty since some of these diseases can be managed very cost‐effectively using evidence‐based control strategies [26, 29, 30]. HAT is classified as one of the most neglected tropical diseases that exclusively affects poor communities in low‐ and middle‐income countries (LMIC), except those areas where tourists have been reported to have contracted the disease on tour of the affected areas. Because NTDs affect mostly the socially vulnerable populations, there are several ethical implications to consider when planning collection and use of these materials. Detection and treatment of these diseases poses many challenges since most of them present similar clinical symptoms con‐ comitant with variation in response in the affected individual to treatment, and lack of accurate diagnostic tests. Medical research to improve health care faces a major problem in the relatively limited availability of adequately annotated and collected bio‐specimens, primarily due to absence of bio‐banking facilities and associated infrastructure to interrogate the specimen to tease out relevant information. This limitation has adversely impacted the pace of scientific advances and successful exploitation of bio‐specimens. Established functional bio‐banks would surmount this limitation by providing framework for transfer of bio‐specimens (tissues, blood, and body fluids) and related health data for research. The KETRI Cryo‐bank holds significant quantities of samples dating from 1930s to date, which include blood, serum, CSF, tissues, semen from trypanotolerant animals, and both parasite and vector DNA collections. This is in addition to the pan African trypanosome isolates of specific biomedical interest (e.g., drug resistance and virulence) from human and nonhuman primates, and livestock. There is therefore great need to collect and store biological materials for research in order to monitor our ecosystems including new and emerging diseases, generate evidence to inform policy, and in the development of mitigation strategies. In the area of human and veterinary medicine, these new and reemerging diseases and conditions have complicated the search for new remedies for their management in the absence of well collected and cryopreserved biological specimens.

#### **10.1. The challenges**

**3.** Clone

20 Cryopreservation in Eukaryotes

**4.** Line

**5.** Population

**6.** Primary isolate

**7.** Sample

**8.** Stabilate

**9.** Stock

**10.** Metacyclics

**11.** Procyclics

**10. Conclusion**

another line of that nature, etc.

several species and subspecies.

This is a population that has been developed from a single trypanosome.

That part of trypanosome population collected on a unique occasion.

A population derived by serial passage *in vivo* or *in vitro* from a primary isolate.

These are the midgut forms of trypanosomes which are found in cultures.

A cryopreserved sample of viable trypanosomes.

are found at the end of transmission cycle.

A laboratory derivative of a stock maintained in different physical conditions, e.g., a species of *T. congolense* maintained in mice only is a line of that nature, while when maintained in cattle only is

The group of trypanosomes present at a given time in a given host or it may consist of a mixture of

This is a stabilate made from a naturally infected host or viable organisms present in a culture or experimental animals following the introduction of the sample from a naturally infected host.

These are the mammalian infective forms of trypanosomes injected by the tsetse fly. Metacyclic forms

Communicable and noncommunicable diseases, including the neglected tropical diseases, cause chronic life‐long disability, hinder economic development, and impair childhood development in resource poor settings in Africa where the diseases are endemic [27, 28]. Control of these diseases could be an efficient way to fight poverty since some of these diseases can be managed very cost‐effectively using evidence‐based control strategies [26, 29, 30]. HAT is classified as one of the most neglected tropical diseases that exclusively affects poor communities in low‐ and middle‐income countries (LMIC), except those areas where tourists The countries that are heavy burdened by disease also experience high levels of poverty. This situation is compounded by new and reemerging diseases and conditions. Climate change has not only resulted in loss of biodiversity but enabled vectors to infest new areas and change transmission dynamics. Some parasites have changed host seeking behavior with time, becoming either more virulent or chronic in nature. Development of drug resistance and appearance of virulent phenotypes is of great public health concern. Whereas the need exists to collect and preserve these materials for R&D in order to find solutions to these challenges, the cost of sample collection from the field is prohibitive. Sites are usually remote with unpassable roads especially in rainy season when disease transmission is high. Once the materials have been collected and transported to the laboratory, there are high costs related to processing, cryopreservation, and maintenance of the cryo‐bank. These are not the usual areas for investment by our governments due to different priorities. There are also challenges related to communities from which samples are collected, they are usually not involved in the plans to collect the materials thereby excluding them in finding solutions to their problems.

#### **10.2. The opportunities**

The above challenges have created great opportunities for collection and storage of parasites and their vectors for use in the development of vaccines, diagnostic tests, and new medicines applying recent technological advances. In the recent past, improvements have been made on the conventional nitrogen freezers through development and adoption of validated methods including a wide range of stem cells. Many cryopreservation protocols exist for freezing and storing various biological materials. These need to be reviewed and tailored toward delivering quality biological materials to our research institutions and products to our clinics. Modalities for sharing of materials by different institutions need to be developed and made operational in order not to disadvantage communities from which the materials are collected and the institutions that have collected, preserved, and maintained these materials in resource‐ constrained settings. The contractual arrangements surrounding areas of the material transfer agreements should be carefully negotiated. National and international institutions (local and foreign), should invest in adequate bio‐specimen management, legal and administrative skills, just as they do for developing scientific skills to facilitate sharing of samples and information associated with the bio‐specimens.

#### **10.3. Lessons learnt: how to establish and sustain cryobank in a resource‐constrained setting**

Collection and cryopreservation of biological materials is critical to research and development but expensive to collect, process, store, and maintain. Institutional top management leadership supported by the existing national, regional and international guidelines, rules and regulations are necessary in providing policy direction and resources [31]. In Kenya, tsetse flies, vectors of human and animal trypanosomiasis, infest mainly conservation areas and wildlife are carriers of pathogens, hence there is a need to work closely with the Kenya Wildlife Service. Through effective collaborations and multidisciplinary approach, it is possible to leverage on all activities undertaken by collaborating institutions to make collections. From the resource‐ constrained perspective, one does not need state‐of‐the‐art bio‐repository to initiate collec‐ tions. Strategic leadership is the key in spearheading:

	- **1.** to define roles and responsibilities for collaborative arrangements among institutions, strictly observing existing rules and regulations
	- **2.** to ensure that communities from which the materials are collected are not taken advantage of
	- **3.** to facilitate the establishment of relevant multidisciplinary teams that cut across several sectors, e.g., human health, livestock, and wildlife
	- **4.** to ensure proper collection, storage, and maintenance of the materials according to the legal mandates of participating institutions
	- **5.** to ensure equity in sharing of the resources,

Effective coordination of field teams is critical as many of the areas from where the collec‐ tions are made are remote with no electricity. Due to the rough terrain and impassable roads, especially during the wet season and when the disease transmission is high, a lot of liquid nitrogen may be lost. In view of this, it is important to ensure adequate liquid nitro‐ gen is available to last the period of the field trip. This is critical and assures viability of the parasites from the remote field sites to the laboratories for preparation and permanent stor‐ age.

In conclusion, it is possible for institutions to collect, process, store, and maintain biological resources according to their legal mandates in resource‐constrained settings. This is only possible through strategic leadership that recognizes the importance of these biological materials to the respective countries and communities from which they are collected. And for organizations requesting for these materials to recognize the efforts and cost of the collection, storage, and maintenance and follow the national and internationally recognized guidelines, rules and regulations regarding the sharing of the same.
