X-Message-Number: 2184 Newsgroups: sci.cryonics From: (Christopher Michael Rasch) Subject: C. elegans freezing protocol. Please comment Message-ID: <> Date: Fri, 30 Apr 93 18:50:41 GMT Hi-- Appended is the protocol I submitted for my Bio44Y independent project. Any suggestions for improvement would be greatly appreciated. Thanks to Nick Szabo for pointing me to the Drosophila embryo research, and to Mike Darwin, for his advice on experimental design. Effects of cooling rate on the survival of L4 and adult stage C. elegans after freezing/vitrification. A number of organisms can survive temperatures below -35 degree C, with up 65% of their total body water as ice. Among these animals include the woolly bear caterpillars (Gynaephora groenlandica), which may spend 10 months of the year frozen solid at temperatures below -50 degrees C, and the striped chorus frog (Pseudacris triseriata). These animals manufacture cryoprotectants (in the case of frogs, glucose) which minimizes freezing damage. (Storey, 1990) This experiment is designed to see how well the nematode C. elegans can survive freezing/vitrification down to liquid nitrogen temperatures. We will compare slow cooling with flash freezing in liquid nitrogn. Since Riga and Webster report that juvenile nematodes survive better than adult nematodes we will also compare survival rates of juvenile and adult nematodes. (Riga, Webster 1991). C.elegans, is a simple 1 mm long worm. It has been extensively studied--all 302 neurons have been mapped at the electron microscope level. The complete cell lineages of the 1000 somatic cells is known, as is a nearly complete genetic map. (Rankin, 1990, pg. 89) C. elegans is capable of non- associative learning; it becomes habituated to successive patterns of taps on its dish. (Mah and Rankin, 1992). The nematode feeds on E. coli, and can be raised in a agar medium. Thus C. elegans is a good potential model for detailed study of freezing on living tissue. The survival of frozen cells critical depends upon the (1) avoidance of intracellular ice and (ii) the presence of intracellular molar concentrations of protective solutes. (Mazur, 1992) Cold tolerant organisms manufacture compounds (often glucose) which minimize freezing damage. As cells cool, intracellular water flows across the cell membrane because of osmotic and vapor pressure gradients. This results in cell shrinkage. Glycerol, the cryoprotectant we'll be using in this experiment, helps minimize shrinking by reducing the mole fraction of other solutes remaining in the nonfrozen water. Glycerol inhibits the formation of crystalline ice, and thus depresses the freezing point of the water. It may also prevent protein denaturation by hydrogen binding with bound water. Other cryoprotectants (DMSO4, ethylen glycol, etc) work in a similar manner. As cells cool, solvent water converts to extracellular ice, and the increasing extracellular concentration of nonpermeating electrolyte or nonelectrolytes damages the cell. When treated with a cryoprotectant, cells don't reach the salt concentrations of nontreated cells until they reach much lower temperatures. Chemical reactions proceed very slowly at such low temperatures and consequently cellular damage is minimized. (Karow, 1981) Materials and Methods Thermometer, range (-200 degrees C/30 degrees C) 20 glass slides w/cover slips filter paper 50 Pasteur pipettes w/bulbs 2 M glycerol solution 2 quarts LN2 (liquid nitrogen) 20 10 mL test tubes LN2 containers (2 1 quart thermos bottles and/or Dewar flasks) microwave liquid Agar medium E. coli bacteria w/media 10 100 CC syringes dissecting microscope freezer (space) (-70 degrees C) test tube tongs gloves C. Elegans hatch approximately 14 hours after fertilization. C.Elegans larvae subsequently develop through four stages, L1, L2, L3, and L4. We will grow a colony of nematodes in the liquid Agar medium; the nematodes feed upon E. coli bacteria. Mazur reports that Drosophila embryos survive 2.1 M concentrations of cryoprotectants at room temperature.(Mazur, 1992). Consequently, we will add the glycerol at 22 degrees C. We will transport one group of 20 L4 nematodes to a 10 ml test tube containing 2 M glycerol; a second group of 20 adult nematodes will be transported to another test tube containing an identical solution. The nematodes will bathe in the solution for 20 minutes, to ensure that glycerol sufficiently perfuses. After perfusion, the test tube will be dipped in LN2 (-196 degrees C) for 10 minutes or until vitrification/freezing is apparent. A second group of nematodes prepared as above, will be placed in the freezer and cooled by ~1 degrees C/minute until they reach -70 degrees C. At that point they will be transferred to a container of liquid nitrogen for another 10 minutes. Both groups will be warmed in a microwave until they reach 22 degrees C. At that point, the nematodes will be transferred to a solution containing no glycerol. We will count the number of nematodes before freezing, and the number of surviving nematodes after freezing. A nematode will count as alive if it is still capable of moving. We will compare the number of surviving L4 nematodes with the number of surviving adults. The freezer, syringes, pipettes, filter paper, and glass slides will be used to perform preliminary tests for freezing nematodes. Expected Results We expect that more L4 nematodes will survive than adults because in Riga's experiment "...A significant number of juveniles than adults survived deep freezing." (Riga, Webster, 1991). We also expect that slow freezing will survive better because intracellular water will have to time osmotically travel across the membrane, and will not become frozen as intracellular ice. However, if C. Elegans prove to be chill- sensitive, as Drosophila are, flash freezing may prove more beneficial. However, we will be using a lower molar concentration of cryoprotectant than Mazur used with the Drosophila embryos. (Mazur, 1992). Karow, A M, Pegg, D E, Organ Preservation for Transplantation, 2nd ed, 1981, Marcel Dekker, Inc. pg 161. Mah, K B, Rankin C H, "Analysis of behavioral plasticity in male Caenorhabditis elegans." Behavioral and Neural Biology 58 211-221, 1991. Mazur, P, Cole K W," Cryobiological Preservation of Drosophila Embryos", Science, 258:1932-1935, 1992. Pickup, J, "Seasonal variation in the cold hardiness of three species of free-living Antarctic nematodes" Functional Ecology, 1990 4(2):257-264) Popiel, I; Vasquez, E M, "Cryopreservation of Steinernema carpocapsae and Heterorhabiditis bacteriophora", Journal of Nematology (abstract). 1991 23(4):432-437 Riga, E, Webster, J M "Cryopreservation of the pinewood nematode, Bursaphelenchus", Journal of Nematology (abstract), 1991 23(4):438-440. Rankin, C H, Beck, C B, and Chiba C M, "Caenorhabditis elegans: a new model system for the study of learning and memory" Behavioral Brain Research, 37 (1990) 89-92 Storey, K B, Storey J M "Frozen and Alive", Scientific American, December 1990, pg. 92-97. Wood, W B ed., _The Nematode Caenorhabditis Elegans_, 1988, Cold Spring Harbor Laboratory. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=2184