CPMB-5 Cryoprotective SolutionDMSO?????????????????????????????????????????????????????????????????????????????????????????????????? 1.0 ml
2.5 M Sucrose ?????????????????????????????????????????????????? 0.8 ml
L-Cysteine/Ascorbic Acid Solution???????????????????????? 0.2 ml
CPMB-2 Base Solution??????????????????????????????????? 6.0 ml
Heat-inactivated bovine serum????????????????????? 2.0 ml
CPMB #2 Basal Solution
Casein Digest Peptone (BBL)????????????????????? 40.0 g
Yeast Extract????????????????????????????????????????????????????????????????????????????? 20.0 g
K2HPO4??????????????????????????????????????????????????????????????? 1.0 g
KH2PO4??????????????????????????????????????????????????????????????? 0.6 g?????
NaCl??????????????????????????????????????????????????????? ??????????? 2.0 g
Distilled water??????????????????????????????????????????????????? 1.0 L
(Autoclave the solution)
L-Cysteine/Ascorbic Acid Solution
L-Cysteine-HCL??????????????????????????????????????????????? 1.0 g
Acorbic Acid?????????????????????????????????????????????????????? 0.1 g
Distilled water????????????????????????????????????????????????? 10.0 ml
Add 9.0 ml of distilled water to a 20 ml beaker and dissolve the first two components.? While stirring, adjust the pH to 7.2 with 10N NaOH (approximately 0.7 ml).? Adjust final volume to 10 ml with distilled water and filter sterilize.
1.?? Harvest cells from several cultures which are in the late logarithmic to early stationary phase of growth.? Place culture vessels on ice for 10 min.
2.?? Invert tubes 20 times and centrifuge at 200 x g for 5 min.????????
3.?? While cells are centrifuging, prepare the cryoprotective solution.?
a)? Place the DMSO in a 16 x 125 mm screw-capped tube and ice until solidified.
b)? Add 0.8 ml of? the 2.5 M Sucrose solution, remove from ice and invert until the DMSO is liquefied.? Return to ice bath.
c) Add 0.2 ml of the L-Cysteine/Ascorbic Acid solution to the DMSO solution and mix.
d)? Add 6.0 ml of the CPMB #2 Basal solution and mix.
e)? Add 2.0 ml heat-inactivated bovine serum and mix.
4.?? Resuspend the cell pellets and pool to a final volume of approximately 10 ml with the supernatant.? Make a determination of the cell density and adjust the concentration of the cells between 5 x 105/ml - 1 x 106/ml using fresh medium.? If the cell concentration is below 5 x 106/ml, centrifuge the cell suspension and resuspend the pellet in a volume the will yield the desired concentration.
5.?? After the cell concentration is adjusted, centrifuge as in step 2.
6.?? Remove as much supernatant as possible and determine the volume removed.
7. ? Resuspend the cell pellet with a volume of the cryoprotective solution equal to the volume of? the supernatant removed.? Invert the tube several times to obtain a uniform cell density.
8.?? Dispense 0.5 ml aliquots into 1.0 - 2.0 ml plastic sterile cryules (special plastic vials for cryopreservation).
9.?? Place vials in a controlled rate freezing unit.? From room temperature cool at -1°C/min to -40°C.? If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.? At -40°C plunge ampules into liquid nitrogen.
10.Store ampules in a liquid nitrogen refrigerator until needed.
11.To establish a culture from the frozen state, place an ampule in a 35°C water bath, until thawed (2-3 min).? Immerse the vial just sufficient to cover the frozen material.? Do not agitate the ampule.
12.Transfer contents of thawed ampule to a 16 x 125 mm screw-capped test tube containing 13 ml of ATCC medium 1978.
13.Screw cap on tightly and incubate at a 15° horizontal slant at 25°C.? Observe the culture daily and transfer when many trophozoites are observed.
