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1. Harvest cells from a culture that is at or near peak density by centrifugation at 800-900 x g for 5 min. Pool the cell pellets into a single tube.
2. Adjust the concentration of cells to 2.0 x 10⁷/mL.  If the concentration is too low, centrifuge at 800-900 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows:  Add the required volume of DMSO to a glass screw-capped test tube and place on ice.  Allow the DMSO to solidify.  Add the required volume of refrigerated ATCC medium 1034.  Dissolve the DMSO by inverting several times.  If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁷ and 7.5% (v/v) DMSO.  The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 60 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place the vials in a controlled rate freezing unit.  Use the following cooling cycle: From room temperature cool at -10°C/min to the heat of fusion; from the heat of fusion to -40°C, cool at -1°C/min.  At -40°C plunge into liquid nitrogen.  The cooling cycle should be initiated no less than 15 and no more than 30 minutes after the addition of DMSO to the cell preparation.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial enough to cover only the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the entire contents of the vial and transfer to a T-25 flask containing 10 mL ATCC Medium 1034 with serum increased to 30%.
10. Incubate the flask horizontally at 25°C with the cap screwed on tightly.

Owczarzak A, et al. The destruction of Schistosoma mansoni mother sporocysts in vitro by amoebae isolated from Biomphalaria glabrata: an ultrastructural study. J. Invertebr. Pathol. 35: 26-33, 1980. PubMed: 7365267

Stibbs HH, et al. Schistosome sporocyst-killing amoebae isolated from Biomphalaria glabrata. J. Invertebr. Pathol. 33: 159-170, 1979. PubMed: 501126

Hertel LA, et al. The symbiont Capsaspora owczarzaki, nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea. Int. J. Parasitol. 32: 1183-1191, 2002. PubMed: 12117501

Amaral-Zettler LA, et al. The Nucleariid Amoebae: More Protists at the Animal-Fungal Boundary. J Eukaryot Microbiol. 48:293-297, 2001. PubMed: 11411837.

Ruiz-Trillo, I, et al. Insights into the evolutionary origin and genome architecture of the unicellular opisthokonts Capsaspora owczarzaki and Sphaeroforma arctica. J Eukaryot Microbiol. 53:379-384, 2006. PubMed: 16968456.

Suga H, et al. The Capsaspora genome reveals a complex unicellular prehistory of animals. Nat Commun 4: 2325, 2013. PubMed: 23942320

Sebé-Pedrós A, et al. Regulated aggregative multicellularity in a close unicellular relative of metazoa. eLife 2: e01287, 2013.

Hertel LA, et al. The symbiont Capsaspora owczarzaki, nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea. Int. J. Parasitol. 32: 1183-1191, 2002. PubMed: 12117501

Nucleotide (GenBank) : ACFS01000000 Capsaspora owczarzaki ATCC 30864, whole genome shotgun sequencing project.