Tuesday, July 24, 2007

Laboratory Techniques

Hi all, Royston here. How are all of you at work? Sorry for not posting before the weekends, because I left my thumbdrive in school locker. For these past 4 weeks, I have been doing literature searchs, protocol write-up as well as subculturing Human Umbilical Vein Endothelial Cells (HUVECs) for my major project.

From the literature searchs, I have learnt that atherosclerosis is actually a chronic inflammatory disease. Cell – cell interaction between circulating monocytes and vascular endothelial cells is a key event in the initiation of atherogenesis. Recent studies have shown endothelial cells treated with soy proteins of isoflavone class inhibited monocyte adhesion, thereby suggesting the athero-protective effects of soy proteins.

I have written protocols of cryopreservation, feeding, and gelatin coating for culture dishes, subculturing and thawing of cells. Protocols are required to minimize the occurrence for errors as well as for trouble-shooting purposes.

Human Umbilical Vein Endothelial Cells (HUVECs) used for my major project, were purchased from Amercian Type Cell Collection. The main advantage of using HUVECs to study endothelial biology is the wide availability of the umbilical cord, a relatively simple method of isolation and the general purity of the cell population obtained. It is important to note that, as with isolation of any cells from a human tissue, there is a potential of risk of infection. Precautions for working with human tissues, such as wearing gloves, a laboratory coat, and safety goggles, must be used at all times. Working in a Biosafety Laboratory level 2 (BSL-2) is also required. This laboratory is negatively pressurized by a vacuum with an ante room in front of the lab, in which these 2 features do not allow any contaminated air from leaving the lab.

1 vial containing 1 mL of HUVECs was thawed and plated on 100-mm 0.2% gelatin coated dishes in commercially prepared HUVEC medium. After 1 day of incubation, media change is done to dilute the traces of DMSO, which is toxic to cells, used in cell freezing media during cryopreservation. After 2 days of incubation, feeding (media renewal) is done to replenish the growth factors and supplements in the media as well as removing any metabolic waste or dead cells that may be inhibitory to cell growth.


However, the picture above shows the cells morphology before feeding, the cells shows severe signs of deterioration which includes granularity around nucleus, cytoplasmic vacuolation and rounding up of cells with detachment from sustrate.


Based on the picture above, it can be seen that all the cells have become unhealthy as are fractile particles present within cells. Cells have slow cell proliferation. Troubleshooting is done to determine the cause of the problem and improvise solutions to solve the problem. Cause was identified as seeding density too low. When HUVECs are set up at too low a density, there is minimum cell-cell interaction between cells. This results in formation of unhealthy cells and slow proliferation. By passaging the culture frequently in new fresh media will solve the problem. This causes the unhealthy cells to die off eventually while new healthy cells will be generated via cell division in fresh medium with abundant cell growth factors.


Picture above shows cells after 3 days of incubation in fresh medium, another round of feeding is required to constantly provide growth supplements for continuous cell growth. The cells are actively dividing and proliferating. A subculture is required once confluence of the dish is reached.

Royston Tan
0503289A
TG01

6 comments:

The Lab Freaks said...

seems that although u appear very free in the lab, u done alot of research ah..
anyway..how often do u have to renew your gelatin coated plates?and why don't u just culture the cells in the flask instead?

Charmaine
TG01

The Lab Freaks said...

what is isoflavone class?

Xuefang

J.A.M.M.Y.S said...

how do you do troubleshooting?

Shahirah
TG01

The Lab Freaks said...

To Charmaine,

I am very 'busy' at work lor.. Gelatin coated plates do not have to be renewed unless the cells somehow do not adhere onto the plates and of course during each subculturing, a newly coated plate have to be used each time. HUVECs can also be cultured using flasks provided they are gelatin-coated before using as gelatin are attachment factor for the cells. And also the CS-C media used for culturing HUVECs is formulated for 37oC and 5% CO2 in the incubator for adjustment of pH. Therefore there is a slight advantage when using a dish will allow better contact with the atmosphere inside the incubator.

To Xuefang,

Isoflavones are secondary metabolites derived from phenylpropanoid metabolic pathway of plant. They are most commonly found in soybean; the major isoflavones in soybean are genistein and daidzein which now believed to have health benefits against cancer and heart disease. Plants use isoflavones and their derivatives to ward off disease-causing pathogenic fungi and other microbes.

To Shahirah,

I have done troubleshooting like culturing the cells onto culture dishes of different sizes and found out that the cells grow better and faster in small dishes due to increase in cell - cell interactions required for proliferation of the cells. And also by adjusting certain media components such as cell growth factor, cell attachment factor, fetal bovine serum concentration and basal media volume used, i am able to solve problems like presence of unhealthy cells and signs of deterioration in the cell cultures.

Thank you for all your questions. Hope you all understand my explanations. Please feel free to ask if you still have any doubts.

Royston
TG01

The Lab Freaks said...

sorry for asking late!
erm, may i ask why the dishes used must be of 0.2% gelatin coated?
Does gelatin help in the growth of the cells or something?

Natalie
TG01 (:

The Lab Freaks said...

Hi Nat,

The gelatin are cell attachment factor required by the cells to be attached to the culture dishes. HUVECs are of adherent cell type, which means they requires attachment before they are able to proliferate actively. Therefore, gelatin aids in the proliferation of cells.

Royston
TG01