This is to confirm that the project entitled “Cryopreservation of human whole blood and monitoring the viability of mononuclear cells” is a legitimate piece of work carried out under my supervision as part of the work required for the completion of the bachelor's degree in BIOMEDICAL Engineering at National Institute of Technology and to the best of my knowledge, this thesis or any part of this thesis has not been submitted to any university/institute/college to the best of my knowledge and for all purposes. I express my sincere and heartfelt gratitude to Prof. Krishna Pramanik. Without her, my lifetime project would not have been possible. I am especially grateful to her for giving me the autonomy to choose the project and autonomy over several other things, while at the same time controlling me, just like the way a kite is controlled by the master after being given freedom to fly high touch the clouds.
By making my foray into cryopreservation, I knew I would join the ranks of people taking small steps toward a dream that would culminate in the proverbial giant leap for humanity. Nowadays, cryopreservation of whole blood and whole blood-derived mononuclear cells (MNCs) has emerged as a promising technique for the cure of subjects going through various blood-related disorders. The present work examined the outputs using a selected cryoprotectant on post-thaw viability mononuclear cells (MNCs) derived from peripheral whole blood during the cryopreservation process, along with preserving peripheral whole blood alone.
SEM and phase contrast microscopy confirm the normal cell morphology of the post-thaw cultured whole blood and mononuclear cells, respectively. On a sultry afternoon, one of my friends who was also assigned under her informed me that she would like to meet me in her spare time to discuss our project in detail. She is the Dean (Student Affairs) is very busy, but she is always there for her students. I went to her room at a convenient time as I approached her room. I felt a tremor of shame because (a) I was the apprentice of one of the best profs in India there is in Bioengineering (b) I was a newbie, didn't expect to live up to her expectation and meet the challenge. I myself was wiped out about the whole thing. Finally when I walked into her room we had a sit down talk, she gave a whole bunch of ideas but deep down she didn't want me to go along with any of those ideas unless I really wanted to go ahead with them. She knew that if I didn't like my project, my input would be low and consequently the output would fall and I would see the research as a task, as a routine, as a punishment. So she encouraged me to find out what I was interested in and she gave me time to set my priorities straight. She told me if I came up with any idea which is possible under the umbrella of NIT Rourkela I have her full support. She asked me to read different research papers talk to my friends and I still don't realize she is always there to help me. Now, one morning after doing pranayama, I was sitting alone in my room when someone played a song that I had heard in my freshman year. Moreover, a flashback comes to me and answers the impending question. During my freshman year we did I didn't have many clubs like we have now, mostly we had social clubs but they were infectiously contagious to the freshmen who wanted to revolutionize people's lives. I receive a phone call from a person who says that he needs it urgently because of involvement in social clubs. a unit of O(-) blood group. I immediately rush down to my wardens office and tell him that a certain gentleman called and requested a unit of O(-) blood type. just figureheads. He opens his computer and a hard copy of about 8-9 pages comes out. He staples it and hands it to me. I had to blink twice when I went through each paper because I couldn't read any O( -) Finally, after going through the sheaf, I found two freshmen who had it. With great difficulty I tried to locate their room numbers and telephone numbers. I knocked at the first guys room when I asked him to donate his blood he was scared to no end .I did a text book on him as instructed by our warden Prof KR Patel but it was in vain. He was a cop out, so donating blood was an absolute no no for him. The second guy was a pushover at first, although I was scared. his.
Cryopreservation refers to the storage of living biological system at very low temperatures (-196⁰c) in (liquid nitrogen) for a period of time so that it can be repaired and returned to the same living state as before.
The main disadvantage of this method is that the cell can only survive for short periods, so the cells cannot be kept for longer periods of time, moreover, there is always the risk of infection through bacterial, viral and other sources. After a while the cells start to lose their viability and this method may not be effective for all cell lines. 1,2}(Adams 1996; Gheorghiu, Lagranderie et al. 1996) It is a preservation process that uses vacuum freezing, drying, heating and low pressure.
The substance to be freeze-dried is first frozen and then the ambient pressure is lowered and heating is done as a result of which the frozen water changes from solid to gas (sublimation). The process of freeze-drying process is also called freeze-drying or freeze-drying. It is necessary to lower the partial pressure of water above the triple point to detect the direct change of ice to vapor and to stop the melting of the sample. The fluid must pass through (Tg). Tg) is the glass transition temperature for non-crystalline substances below which the substance behaves as glass (hard, brittle) and above which it behaves like rubber (can bend).
Vitrification occurs with water if it contains a cryoprotectant that inhibits the formation of ice crystals. Note: The main drawback of using vitrification for preservation is the toxicity caused by the higher concn.
LITERATURE REVIEW
Rapid cooling rate causes formation of intracellular ice crystals which are harmful to cells and cause mechanical damage. In this scenario, the role of the intracellular cryoprotectant, as discussed earlier, becomes very significant. While at slow cooling rate there is formation of osmotic difference due to different solution conditions and generation of extracellular ice, these factors can damage cells by diffusion of water outside cells and cells can shrink and lead to osmotic dehydration. A slow cooling causes freezing of the extracellular fluid, while intracellular fluid remains unfrozen for a while.
This results in an increase in the concentration of salts in the extracellular solution, which causes cellular dehydration and osmotic flow through the cell membrane. Schematic diagram showing when cells cool rapidly, intracellular ice formation occurs and when extracellular ice formation occurs slowly.
Therefore the key is to optimize the cooling process
Cryoprotection is essentially multiple loading of cells and tissues with cryoprotectants in high doses, which allows water to pass through osmosis and solutes to move by diffusion. Freezing means the formation of ice and the reduction of water and the increase of solute, thereby establishing osmosis and diffusion. Membranes, being selectively permeable, react in different ways depending on the solute, sometimes allowing diffusion, sometimes not. If and are the internal and external osmolarity and A is the area of the membrane, then.
20}(Kedem and Katchalsky 1958) Kadem and Katchalsky took into account that the solute and solvent use a common gateway to enter the membrane and modified the 3rd and 4th equations by using the solute and solvent communication called the reflection coefficient. Now the new equation becomes . - )].5.
Cyopreservation of human whole blood
Protocols for studying Human whole blood under SEM
Centrifuge separation of MNC protocols
Cooling plots of MNC
TRYPAN BLUE STAINING PROTOCOLS{25}) (Shapiro and Leif 2003) Material needed
RESULTS AND DISCUSSION
RESULTS
Hydroxyethyl starch provides the best viability for MNCs, while dextran is the least and DMSO performs an intermediate work after cryopreservation in liquid nitrogen.
CONCLUSION
34; A look at the field of freeze-drying: classic issues and new ventures. "Drugs and the Pharmaceutical Sciences 96:1-30. 34; Living in a frozen state: adaptive strategies for natural freezing tolerance in amphibians and reptiles." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 258(3): R559. 34;Guidelines for the diagnosis and treatment of iron deficiency and anemia in inflammatory bowel diseases." Inflammatory bowel diseases.
34;Recent Developments and Concepts in Sperm Cryopreservation and Evaluation of Their Post-Thaw Function." Reproduction, Fertility and Development. 34;Mechanisms of Cryoinjury in Living Cells." ILAR/National Research Council Journal, Institute of Laboratory Animal Resources 41(4): 187. 34; Freezing Living Cells: Mechanisms and Implications." American Journal of Physiology-Cell Physiology 247(3): C125. Dan 2007) Cryopreservation and Lyophilization Protocols.
INSTRUMENTS USED FOR THE EXPERIMENTS
