Process Of Cryopreservation And Vitrification Biology Essay

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INTRODUCTION

In the field of Biotechnology there is a field name Cryobiology (Study of living things in low temperature).In this part of subject we preserved cells, tissues, gametes, embryos, and even organs (Heart, kidney, liver etc.) under hypothermic conditions .

In Cryobiology there are four major areas of study.

Cold adaptation

Lyophilization (freeze drying)

Cryosurgery

Physics of ice nucleation and heat transfer

Cryopreservation

In the field of cryobiology, cryopreservation takes place. In this the viable cells are preserved for the time that we want to take as for as the long time or for the short time in very low temp with the help of the liquid nitrogen. Chris Poldge (1926-2004) preserves the viable cells (sperm) first time in the history of science. In cryopreservation there are three major applications:-

Biomedicine

Animal reproduction

Conservation

Biomedicine

It is the science in which biology, chemistry and physics involved and applies biologically to clinical practice. In this the molecular interaction takes place for understanding the vivo level. Human sperm, egg cell and embryos are cryopreserved for the infertility of the couples that are unable to produce the beneficial cells for the birth of their young ones. By this many babies are born in which Loise brownies the first test tube baby in 1978.

Animal reproduction

Some of the animals who are in the category of dangered species are preserved by the help of cryopreservation. By preserving the semen, gametes and embryos of various domestic and non-domestic animals so that we can conserve the livestock genetic for the interest of international community.

Conservation

"Means preservation of products". In cryopreservation the conservation of gametes, embryo of the animals and plants in the cryobanks and seed banks respectively.

Process of cryopreservation

In the process of cryopreservation two injuries occur when in low temperature first is:-

Freezing injury.

Chilling injury.

Vitrification

Is the conservation of certain biological things at extremely low temperature without any freezing. As in this formation of glassy or amorphous solid state appears which didn't form ice crystals.Vitrification is a rapid process as compare to cryopreservation.

Materials and Methods

Material Used for Cryopreservation method and Vitrification method

1 tube containing zebra fish ovarian follicles.

1 tube containing 50% L-15 medium, ph: 7.2.

1 tube containing 4 M DMSO in 50% L-15 medium.

1 tube containing 8 M methanol in 50% L-15 medium.

1 tube containing Trypan Blue solution.

Tricane (0.6 mg/ml)

1 forcep and 1 knife.

Cryo-straws (0.5 and0.25 ml).

1 Cryo - loop.

Micro pipettes and pipette tips (1 and 20 µl).

2 microscope slides and coverslips.

6 wells culture culture plate.

1 pair of nitrile gloves and apron.

Here M = Molar

DMSO = Di methyl sulpho oxide

L-15 medium = Leibovitz medium

Methanol = Cryoprotectant

Tricane = Anaesthetsed solution

Techniques

First we prepare the solution of different molarity in the test tubes by adding suitable 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 gloves and then we take a female zebra fish and put in the tricane(0.6mg/ml) for 5min so that it get anaesthetized. When it is fully anaesthetized cut the head of the fish and then picks up with the forcep and cut the fish from the abdomen part to tail horizontally before the ovaries are removed. Now place the ovaries in the petridish containing L-15 medium at 22ËšC immediately. Oocytes will be separated by using forceps and scissors.Trypan Blue will be used to assess membrane integrity TB.Then the ovarian follicles are extracted through the ovaries. These ovarian follicles are now put in the 8 M solution of methanol 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 then wash them in L-15 medium now take the slide and place it under the microscope, we observe that the unstained or transparent follicles are viable whereas stained blue are non-viable as the experiment done in the lab.

Controlled Slow Cooling

It is the one of the main process of this part. In this cryoprotectant is used for the protection of cells from the freezing of the cells, tissues, embryo etc. so that the damage to these biological tissues not occurs.4 M Methanol solution will be used in which 50% Leibovitz L-15 medium is dissolved and here methanol is act as cryoprotectant.In this ovarian follicles are put in 4 M methanol sol with 50% L-15 solution for half an hour to incubate at 22ËšC.Now after 30 mins load ovarian follicles into 0.5ml plastic straws and put into a programmable cooler. By following the protocol cooling at 5ËšC/min from 20ËšC to -12.5ËšC, the seeding temperature (The stage in which the manual cooling is done).When the temperature decreases to -12.5ËšC manual seeded is done and temp goes to -80ËšC for 10 min, now by taking out the plastic straws containing ovarian follicles of zebra fish are then plunged in liquid nitrogen at -196ËšC and remain it for 10 min.

Thawing

(It is the process in which solid state changes to liquid state by losing its stiffness, numbness or impermeability by being warmed).Thaw the samples using a water bath at 27ËšC .The plastic straw pipe containing the follicles are now take out from the water bath and place it in the petridish.

Now take out the follicles in the petridish which are in 4 M methanol. Remove the cryoprotectant in four steps by decreasing the molarity of the methanol to 2M, 1M, and 0.5M in L 15 medium for 2.5 min respectively. Then transfer the ovarian follicles in the slide and transfer it in the L-15 medium.BY assessing 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 process to test the ability of it in the process. 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 unsealed 0.25ml plastic straws with the syringe at room temperature or on to the cryo loop (5-10 µl) using a 20µl pipette.

Put the loaded straws directly into liquid nitrogen whereas the cryo loop are placed on the surface of a vitrification block immerged into the liquid solution. After few mins transparent glassy appearance indicates that the solution is vertified and milky appearance indicates de-vitrification.

Process

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 doing this transfer the follicles in 3M,6Mor8M DMSO for 5 min and then after some time load these into the unsealed 0.25ml plastic straws with the syringe very gently or by cryo-loop(5µl) by a 20µl pipette.

Dip the loaded straws and cryo-loop in the liquid nitrogen directly for some time.

Thawing

Transfer the straw to a water bath at 27ËšC .By doing this, material come to liquid state then immerse the loaded cryo-loop into L-15 at 22ËšC.

Dilute cryoprotectants gradually in four steps (2M, 1M and 0.5M DMSO in 50% L-15 medium for 2.5 min for each step),before final transfer to ovarian follicles to L-15 medium.Assest viability by Trypan Blue staining.

Result

Analysed Data for Slow cooling (Cryopreservation)

S.no

Concentration of L-15 solutiom

Molar conc.of Methanol

No.of Oocytes

Time to incubate

Viability %

1

50%

8M

30

30 Min

5%

2

50%

4M

30

30Min

8%

3

50%

2M

30

30 Min

12%

4

50%

1M

30

30Min

14%

5

50%

0.5M

30

30 Min

20%

Here Methanol is used as a cryoprotectant.

Viability of Oocytes will be calculated by the formula:

Viability% = Number of viable oocytes X 100%

Total number of oocytes

= 6 X 100

30

Viability% = 20%

Data analysis in Vitrification

S.No

L-15 Medium

Molar Conc. Of DMSO

No.of Oocytes

Time to Incubate

Temp.

1

50%

4M

30

10Min

Room Temp.

2

50%

2M

30

10Min

Room Temp.

3

50%

1M

30

10Min

Room Temp.

4

50%

0.5M

30

10Min

Room Temp.

Here DMSO is used as a cryoprotectant

Viability of Oocytes will be calculated by the formula:

Viability% = Number of viable oocytes X 100%

Total number of oocytes

= 12 X 100

30

Viability% = 40%

So,

Viability % of oocytes after slow cooling = 20%

Viability %of oocytes after Vitrification = 40%

Graph showing slow cooling

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Discussion

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