**3. Cryopreservation**

#### **3.1. Cryopreservation media**

The most commonly used cryopreservative is 20% glycerol in EDTA saline glucose (ESG). ESG is first prepared by dissolving 8.00 g NaCl, 0.30 g KH2PO4, 2 g EDTA (disodium or di‐ potassium), and 2 g glucose in 800 ml distilled water, adjusting the pH to 8.0, and then top‐ ping up to 1 liter with deionized/distilled water. Glycerol is then added to the prepared solution at a ratio of 1:4 to make 20% glycerol in ESG. Recently, Triladyl® (MiniTüB GmbH and CO. KG, Tiefenbach, Germany), a commercially available culture medium that is tradi‐ tionally used for preserving bull semen, has been found suitable for cryopreservation of try‐ panosomes. Triladyl is most efficient when added to the infected biological fluids/materials to a concentration of 40–90% [18, 19]. Triladyl has subsequently been adopted as an alterna‐ tive cryopreservative at the KETRI Trypanosome Bank.

#### **Buffers commonly used in cryopreservation**

*EDTA saline glucose (ESG) pH 8.0*

NaCl 8.00 g

KH2PO4 0.30 g

EDTA (disodium or dipotassium) 2 g

Glucose 2 g

**Figure 4.** Stabilate storage dewars.

8 Cryopreservation in Eukaryotes

**3. Cryopreservation**

**3.1. Cryopreservation media**

At the first peak of parasitemia between 1.3 × 108

with a trypanosome concentration below 6.3 × 107

trypanosome species does not infect rodents [17].

1.3×108

**2.3. Isolation and propagation of low parasitemia blood samples in laboratory rodents**

When the parasitaemia in the whole blood of the naturally infected host is low i.e. below

ml, the infected mice are euthanized and the blood harvested by cardiac puncture into tubes containing EDTA as anticoagulant. The blood is mixed gently before addition of a cryopre‐ servative at a ratio of 1:1. The samples are suspended in liquid nitrogen vapor for at least 2 h using a cooling jacket before permanent storage in liquid nitrogen at –196°C [15, 16]. Samples

of glycerol to infected biological fluids to a final concentration of 20%, especially if the isolated

The most commonly used cryopreservative is 20% glycerol in EDTA saline glucose (ESG). ESG is first prepared by dissolving 8.00 g NaCl, 0.30 g KH2PO4, 2 g EDTA (disodium or di‐ potassium), and 2 g glucose in 800 ml distilled water, adjusting the pH to 8.0, and then top‐ ping up to 1 liter with deionized/distilled water. Glycerol is then added to the prepared solution at a ratio of 1:4 to make 20% glycerol in ESG. Recently, Triladyl® (MiniTüB GmbH and CO. KG, Tiefenbach, Germany), a commercially available culture medium that is tradi‐ tionally used for preserving bull semen, has been found suitable for cryopreservation of try‐ panosomes. Triladyl is most efficient when added to the infected biological fluids/materials

 trypanosomes/ml, the anticoagulated infected blood sample is intraperitoneally inoculated into an immunosuppressed laboratory rodent. It is advisable to inoculate two rodents, usually mice, per positive blood sample (isolate). This is carried out in the field where the animals are given identification numbers and transported to the laboratory for monitoring. In the laboratory, the infected mice are maintained on commercial mice pellets (Unga Feeds Ltd., Kenya), provided with water *ad libitum* and monitored for development of parasitemia.

trypanosomes/ml and 2.5 × 108 trypanosomes/

/ml may also be preserved by direct addition

Dissolve in about 800 ml distilled water, top up to 1 l.

*Normal saline*

Dissolve 8.5 g NaCl in 1 l of distilled water

*Phosphate Saline glucose (PSG) pH 8.0*

Na2PHO4 5.392 g/l

Na2HPO4 (H2O)12 (hydrous) 13.608 g/l

NaH2PO4 0.239 g/l

NaH2PO4(H2O) hydrous 0.276 g/l

NaH2PO4(H2O)2 hydrous 0.312 g/l

NaCl 1.7 g/l

Glucose 10.00 g

Dissolve in about 800 ml distilled water; top up to 1 l.

#### **3.2. Cryopreservation procedure**

Biological materials from infected vertebrate hosts (blood, cerebrospinal fluid (CSF), and lymph node aspirates) and/or body parts of tsetse fly vectors are processed as previously described [17, 20, 21]. Briefly, samples with a concentration of more than 6.3 × 107 trypano‐ somes/ml are mixed with a cryopreservative (20% glycerol in ESG or 40–90% Triladyl) at a ratio of 1:1. The diluted sample is then loaded into an ampoule and wrapped or held in a cooling jacket which is suspended into the vapor space of a liquid nitrogen shipper (temperature range of –60 to –80°C) for transportation to the laboratory. Samples from infected humans or animals with parasite counts below 6.3 × 107 trypanosomes/ml (equivalent to antilog 7.8) [14], or suspects with a low packed cell volume (usually <25%) are inoculated into laboratory rodents for amplification/propagation [19]. The rodents may be immunosuppressed using either cyclophosphamide at 100 mg/kg daily for three consecutive days or by gamma‐irradiation at 600 rads (6 gray) for 6 min before the inoculation [22, 15]. Swiss white mice are preferred for propagation of most species of trypanosomes, while Mastomys natalensis are preferred for *T. b. gambiense*[23]. Inoculated rodents are sacrificed at the first peak of parasitemia and the blood harvested by cardiac puncture into tubes with EDTA as anticoagulant. The blood is mixed gently before addition of a cryopreservative at a ratio of 1:1. The diluted samples are then suspended in liquid nitrogen vapor for at least 2 h before being stored permanently in liquid nitrogen at –196°C [24, 25].

#### **Essential requirements for cryopreservation**

The following information is collected and recorded:

jacket which is suspended into the vapor space of a liquid nitrogen shipper (temperature range of –60 to –80°C) for transportation to the laboratory. Samples from infected humans or animals

suspects with a low packed cell volume (usually <25%) are inoculated into laboratory rodents for amplification/propagation [19]. The rodents may be immunosuppressed using either cyclophosphamide at 100 mg/kg daily for three consecutive days or by gamma‐irradiation at 600 rads (6 gray) for 6 min before the inoculation [22, 15]. Swiss white mice are preferred for propagation of most species of trypanosomes, while Mastomys natalensis are preferred for *T. b. gambiense*[23]. Inoculated rodents are sacrificed at the first peak of parasitemia and the blood harvested by cardiac puncture into tubes with EDTA as anticoagulant. The blood is mixed gently before addition of a cryopreservative at a ratio of 1:1. The diluted samples are then suspended in liquid nitrogen vapor for at least 2 h before being stored permanently in liquid

**5.** Trypanosomes either from the cryo‐bank or from infected hosts (humans/animals) or tsetse flies

trypanosomes/ml (equivalent to antilog 7.8) [14], or

with parasite counts below 6.3 × 107

10 Cryopreservation in Eukaryotes

nitrogen at –196°C [24, 25].

**1.** Immunosuppressed mice

**2.** Mice pellets

**3.** Sawdust

**6.** Glycerol

**7.** Cotton wool

**8.** Tissue paper

**10.** Plasticine

**9.** Capillary tubes (plain)

**11.** 4.5 ml plastic ampoules

**12.** Liquid nitrogen

**13.** Cooling jacket

**1.** Host of isolation

**Essential requirements for cryopreservation**

**4.** Disposable 1 ml syringes and needles gauge 26

The following information is collected and recorded:

**2.** Locality (georeferenced) and year of isolation

**4.** Number of passages the trypanosomes has undergone before cryopreservation

**3.** Scientist who did the isolation

**8.** Physical location of the sample in the cryo‐bank

In event of cryopreservation from the natural host:

At the KETRI trypanosome cryo‐bank, the first parasite population isolated from the field is the original or primary isolate. If the parasite numbers are high, the primary isolate is cryo‐ preserved as original field isolate; however, in order to sustain/maintain the original cryopre‐ served sample and to produce adequate material for research, the original isolate is expanded in the appropriate animal model and cryopreserved as a derivative of the original sample, but with a different bank reference number from the first passage number. Subsequent passages or derivatives of the same are given different reference numbers in order to monitor the use and performance of the particular isolates. Clones may be prepared either from the primary or subsequent passages or both. All these events are monitored and recorded for future reference and when issuing materials for research.

#### **3.3. Preparation of the cryopreserved sample for the infection of laboratory animals**

Following receipt of the duly signed and approved request form from the scientist, the person incharge of issuance of cryopreserved material records the physical position of the sample in the cryo‐bank. The sample is retrieved from its position, the ampoule cork opened; and using a pair of forceps, the reference number is confirmed. One capillary is removed and transferred into an ampoule placed on ice for thawing. The remaining stabilate is returned into its position in the bank before it thaws.

#### **The procedure for infection of laboratory animals**

Precaution: