Process Of Cryopreservation And Vitrification Biology Essay
In the field of Biotechnology there is a field name Cryobiology ( Study of populating things in low temperature ) .In this portion of topic we preserved cells, tissues, gametes, embryos, and even variety meats ( Heart, kidney, liver etc. ) under hypothermic conditions.In Cryobiology there are four major countries of survey.Cold versionLyophilization ( freeze drying )CryosurgeryPhysicss of ice nucleation and heat transportation
In the field of cryobiology, cryopreservation takes topographic point. In this the feasible cells are preserved for the clip that we want to take every bit for as the long clip or for the short clip in really low temp with the aid of the liquid N.
Chris Poldge ( 1926-2004 ) preserves the feasible cells ( sperm ) first clip in the history of scientific discipline. In cryopreservation there are three major applications: -BiomedicineAnimal reproductionConservation
It is the scientific discipline in which biological science, chemical science and natural philosophies involved and applies biologically to clinical pattern. In this the molecular interaction takes topographic point for understanding the vivo degree. Human sperm, egg cell and embryos are cryopreserved for the sterility of the twosomes that are unable to bring forth the good cells for the birth of their immature 1s. By this many babes are born in which Loise brownies the first trial tubing babe in 1978.
Some of the animate beings who are in the class of dangered species are preserved by the aid of cryopreservation. By continuing the seeds, gametes and embryos of assorted domestic and non-domestic animate beings so that we can conserve the farm animal familial for the involvement of international community.
“ Means saving of merchandises ” .
In cryopreservation the preservation of gametes, embryo of the animate beings and workss in the cryobanks and seed Bankss severally.
Procedure of cryopreservation
In the procedure of cryopreservation two hurts occur when in low temperature foremost is: -Freezing hurt.Chilling hurt.
Is the preservation of certain biological things at highly low temperature without any freeze. As in this formation of glassy or formless solid province appears which did n’t organize ice crystals.Vitrification is a rapid procedure as comparison to cryopreservation.
Materials and Methods
Material Used for Cryopreservation method and Vitrification method
1 tubing incorporating zebra fish ovarian follicles.1 tubing incorporating 50 % L-15 medium, pH: 7.2.1 tubing incorporating 4 M DMSO in 50 % L-15 medium.1 tubing incorporating 8 M methyl alcohol in 50 % L-15 medium.1 tubing incorporating Trypan Blue solution.Tricane ( 0.
6 mg/ml )1 forcep and 1 knife.Cryo-straws ( 0.5 and0.25 milliliter ) .
1 Cryo – cringle.Micro pipettes and pipette tips ( 1 and 20 Aµl ) .2 microscope slides and coverslips.
6 Wellss civilization civilization home base.1 brace of nitrile baseball mitts and apron.Here M = MolarDMSO = Di methyl sulpho oxideL-15 medium = Leibovitz mediumMethanol = CryoprotectantTricane = Anaesthetsed solutionTechniquesFirst we prepare the solution of different molar concentration in the trial tubing by adding suited concentration of L-15 in it.
Cryopreservation of zebra fish ovarian follicles
To obtain the ovarian follicles of zebra fish, at first wear the nitrile baseball mitts and so we take a female zebra fish and set in the tricane ( 0.
6mg/ml ) for 5min so that it acquire anaesthetized. When it is to the full anaesthetized cut the caput of the fish and so picks up with the forcep and cut the fish from the venters portion to chase horizontally before the ovaries are removed. Now place the ovaries in the petridish incorporating L-15 medium at 22EsC instantly. Oocytes will be separated by utilizing forceps and scissors.Trypan Blue will be used to measure membrane unity TB.Then the ovarian follicles are extracted through the ovaries. These ovarian follicles are now put in the 8 M solution of methyl alcohol in 50 % L-15 medium.
Now take some of the ovarian follicles in the microscope slides and Incubate ovarian follicles in 0.2 % of Trypan blue at room temp for 3 to 5 min.When the ovarian follicles are incubated so rinse them in L-15 medium now take the slide and topographic point it under the microscope, we observe that the unstained or crystalline follicles are feasible whereas stained bluish are non-viable as the experiment done in the lab.
Controlled Slow Cooling
It is the 1 of the chief procedure of this portion. In this cryoprotectant is used for the protection of cells from the freeze of the cells, tissues, embryo etc.
so that the harm to these biological tissues non occurs.4 M Methanol solution will be used in which 50 % Leibovitz L-15 medium is dissolved and here methyl alcohol is act as cryoprotectant.In this ovarian follicles are put in 4 M methyl alcohol colloidal suspension with 50 % L-15 solution for half an hr to incubate at 22EsC.Now after 30 mins load ovarian follicles into 0.5ml fictile straws and set into a programmable ice chest. By following the protocol chilling at 5EsC/min from 20EsC to -12.
5EsC, the seeding temperature ( The phase in which the manual chilling is done ) .When the temperature decreases to -12.5EsC manual seeded is done and temp goes to -80EsC for 10 min, now by taking out the plastic straws incorporating ovarian follicles of zebra fish are so plunged in liquid N at -196EsC and remain it for 10 min.
( It is the procedure in which solid province alterations to liquid province by losing its stiffness, numbness or impermeableness by being warmed ) .Thaw the samples utilizing a H2O bath at 27EsC.
The plastic straw pipe incorporating the follicles are now take out from the H2O bath and topographic point it in the petridish.Now take out the follicles in the petridish which are in 4 M methyl alcohol. Remove the cryoprotectant in four stairss by diminishing the molar concentration of the methyl alcohol to 2M, 1M, and 0.
5M in L 15 medium for 2.5 min severally. Then reassign the ovarian follicles in the slide and reassign it in the L-15 medium.
BY measuring their viability by Trypan Blue staining.
Vitrification of zebra fish ovarian follicles
Vitrification of solutions
DMSO ( Di methyl sulphoxide ) will be used as cryoprotectant in the vitrification procedure to prove the ability of it in the procedure. In this three different molar concentrations solutions are prepared i.e. 3M, 6M and 8M in 50 % L-15 medium.Load the solution in the uncertain 0.25ml fictile straws with the syringe at room temperature or on to the cryo cringle ( 5-10 Aµl ) utilizing a 20Aµl pipette.Put the laden straws straight into liquid N whereas the cryo cringle are placed on the surface of a vitrification block immerged into the liquid solution.
After few mins crystalline glassy visual aspect indicates that the solution is vertified and milklike visual aspect indicates de-vitrification.
In the petridish there are different concentration of DMSO are present. Take ovarian follicles and incubate it with 2M DMSO in 15 % L-15 medium at room temperature for 10 mins.After making this transportation the follicles in 3M,6Mor8M DMSO for 5 min and so after some clip burden these into the uncertain 0.25ml fictile straws with the syringe really gently or by cryo-loop ( 5Aµl ) by a 20Aµl pipette.Dip the laden straws and cryo-loop in the liquid N straight for some clip.
Transfer the straw to a H2O bath at 27EsC.By making this, material semen to liquid province so immerse the laden cryo-loop into L-15 at 22EsC.Dilute cryoprotectants bit by bit in four stairss ( 2M, 1M and 0.5M DMSO in 50 % L-15 medium for 2.5 min for each measure ) , before concluding transportation to ovarian follicles to L-15 medium.
Assest viability by Trypan Blue staining.
Analysed Data for Slow chilling ( Cryopreservation )
S.noConcentration of L-15 solutiomMolar conc.of MethanolNo.
of OocytesTime to incubateViability %150 %8M3030 Min5 %250 %4M3030Min8 %350 %2M3030 Min12 %450 %1M3030Min14 %550 %0.5M3030 Min20 %Here Methanol is used as a cryoprotectant.Viability of Oocytes will be calculated by the expression:Viability % = Number of feasible oocytes X 100 %Entire figure of oocytes= 6 Tens 10030Viability % = 20 %
Data analysis in Vitrification
S.NoL-15 MediumMolar Conc.
Of DMSONo.of OocytesTime to IncubateTemp.150 %4M3010MinRoom Temp.250 %2M3010MinRoom Temp.350 %1M3010MinRoom Temp.450 %0.5M3010MinRoom Temp.Here DMSO is used as a cryoprotectantViability of Oocytes will be calculated by the expression:Viability % = Number of feasible oocytes X 100 %Entire figure of oocytes= 12 Tens 10030Viability % = 40 %So,Viability % of oocytes after slow chilling = 20 %Viability % of oocytes after Vitrification = 40 %
Graph demoing slow chilling
Degree centigrades: UsersGauravDesktopuu.png