Volume 14, Issue 56 (9-2024)
NCMBJ 2024, 14(56): 69-82 |
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Bani Aqeel H, Fatahi I, Eskoiyan R, Kalani M R. Membrane Washing, a New Method to Isolate Membrane Phospholipids Without Cell Destruction. NCMBJ 2024; 14 (56) :69-82
URL: http://ncmbjpiau.ir/article-1-1703-en.html
URL: http://ncmbjpiau.ir/article-1-1703-en.html
Department of Biology, Ayatollah Amoly Branch, Islamic Azad University, Amol, Iran , esmail_fattahy@yahoo.com
Abstract: (418 Views)
Aim and Background: An important part of medical biology studies is devoted to the production of an efficient non-viral structure for gene transfer. The materials of these structures are expensive and impose a lot of cost on the researcher. Enveloping the DNA molecule in a closed space of a lipid bilayer membrane evokes the most similar structure to a liposome. This study is to investigate the possibility of using phospholipids taken from the membrane of a cell to make liposome and transfer genes or drugs to the same cell.
Material and Methods: Five mammalian (HEK293, Hela, SW480, expi-CHO, CHO) and one yeast (Saccharomyces cerevisiae) cells were selected and cultured in a suitable culture medium. A new membrane washing method was performed with different combinations of non-polar solvents of chloroform, methanol, ammonium hydroxide, …, without breaking and destroying the cells. Purification and separation of membrane phospholipids was done by thin layer chromatography (TLC). The mobile phase solvent was optimized for the best separation of phospholipids.
Results: By comparing six combinations of membrane washing solvents, the mixture of chloroform, methanol, ammonium hydroxide, acetic acid (65:25:5:5) had the best efficiency for separating and extracting phospholipids from the cell membrane. The main phospholipids that make up the membrane were well separated by optimized mobile phase thin layer chromatography. The results of this study showed that in CHO, Expi-CHO and SW480 cells, the amount of Membrane phospholipids from the most to the least include POPC, POPE, POPS, CHOL, SM and IMP. POPS contribution was different for Hela and HEK293 cells. Yeast cells can be an acceptable source of phospholipid for purification due to fast growth and cheap culture medium.
Conclusion: By using a special combination of non-polar solvents, the phospholipids of the cell membrane can be washed. These phospholipids can be well separated by thin layer chromatography using a special mobile phase solvent. The cost of this process is much lower than the preparation of industrial transfection compounds. It seems that gene transfer by liposomes made with natural phospholipids should have a much higher efficiency, which should be studied in future research.
Material and Methods: Five mammalian (HEK293, Hela, SW480, expi-CHO, CHO) and one yeast (Saccharomyces cerevisiae) cells were selected and cultured in a suitable culture medium. A new membrane washing method was performed with different combinations of non-polar solvents of chloroform, methanol, ammonium hydroxide, …, without breaking and destroying the cells. Purification and separation of membrane phospholipids was done by thin layer chromatography (TLC). The mobile phase solvent was optimized for the best separation of phospholipids.
Results: By comparing six combinations of membrane washing solvents, the mixture of chloroform, methanol, ammonium hydroxide, acetic acid (65:25:5:5) had the best efficiency for separating and extracting phospholipids from the cell membrane. The main phospholipids that make up the membrane were well separated by optimized mobile phase thin layer chromatography. The results of this study showed that in CHO, Expi-CHO and SW480 cells, the amount of Membrane phospholipids from the most to the least include POPC, POPE, POPS, CHOL, SM and IMP. POPS contribution was different for Hela and HEK293 cells. Yeast cells can be an acceptable source of phospholipid for purification due to fast growth and cheap culture medium.
Conclusion: By using a special combination of non-polar solvents, the phospholipids of the cell membrane can be washed. These phospholipids can be well separated by thin layer chromatography using a special mobile phase solvent. The cost of this process is much lower than the preparation of industrial transfection compounds. It seems that gene transfer by liposomes made with natural phospholipids should have a much higher efficiency, which should be studied in future research.
Type of Study: Research Article |
Subject:
Cellular and molecular
Received: 2024/12/14 | Accepted: 2024/09/22 | Published: 2024/09/22
Received: 2024/12/14 | Accepted: 2024/09/22 | Published: 2024/09/22
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