Hello! Im back to share with you how to collect corpus cavernosum (CC) tissue, which is this spongy erectile tissue that forms the penis in the male rat, from the rat to culture your own cells!
Firstly, we give rat anaesthesia intraperitoneally and wait for around 5 minutes for the rat to lose its body sensation before starting to dissect the rat.
A perineal incision (the region between the anus and scrotum of the male rat) is done on the rat using a surgical scissors and the opening is held stable using hooks. Then, using the arterial forceps and toothed forceps, we locate the bulbocavernosus muscle, which looks like a small white triangular pouch. The corpus cavernosum is within the pouch and therefore, we have to retrieve the whole pouch by cutting it out using the scissors and toothed forceps. 3 cuts is made, one at the penile stem, the other 2 at the left and right crus to remove the entire bulbocavernosus muscle.
To retrieve the corpus cavernosum from within the pouch, we have to cut away the muscles and tunica albuginea surrounding the corpus cavernosum. The corpus cavernosum is then minced and centrifuged in PBS for washing. PBS was then aspirated and the cell pellet was mixed with DMEM by pipetting. Then the mixture would be added to culture flasks that was pre-loaded with fresh DMEM. The flasks would then be placed in the carbon dioxide incubator for growth.
Renee
TG02
0703634F
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Renee,
ReplyDelete(1) Why is PBS used for washing instead of other solutions? e.g. DI water?
(2) I noticed that your medium used for cell culturing is DMEM. Does your DMEM medium contains other additives e.g. fetal bovine serum (FBS) or ant-fungal/bacteria additives?
(3) What are the incubation conditions? e.g. Temperature? How many percent of carbon dioxide?
Li Yinliang Alex
TG02 0704894E
Group 8
Hi renee!!,
ReplyDeletewhen you give the rat anaesthesia, how do you confirm that the rat is really asleep after 5 mins? If the rat is fatter, do you give a heavier anaesthesia? How much and what type of anaesthesia do you use?
when you get the cells, besides culturing the cells, what else can you do with the cells?
LIM JIA HUI (Joey)
0703605F
TG01 Group 2
I believe that for you to research on something like that using the rat, it must be useful for man-kind. So is the corpus cavernosum (CC) tissue aka spongy erectile tissue of the rat similar to that of the male population of man-kind? If yes, how similar. If no, how different.
ReplyDeleteIn addition, what will be the muscles and tunica albuginea use for? Thrown away? Or will it be used for some other work?
Out of curiosity, what will be the outcome of the rat? To live or to die? What is the use of letting the rat live without an important muscle? Will the rat still be able to mate?
Alvin
16 July 2009
Hello people, here is the reply to the questions!
ReplyDeleteReply to Alex:
1. Firstly, we use phosphate buffer saline because saline is an isotonic fluid and therefore, will not cause the cells to burst when we wash the cells in PBS. D.I water is a hypotonic fluid and therefore, the use of it to wash the cells will cause the cells to burst as therefore would be a net movement of water into the cells.
2. Yups, the medium that I use is reconstituted DMEM, which contains fetal bovine serum and antibiotics such as streptomycin, penicillin G and fungizone.
3. The incubation conditions are the same as in school, which is 5% carbon dioxide at 37 degree celcius.
Reply to Joey:
1. When we give anaesthesia, the rat is usually down by 5 minutes. We check by lying it face-up on the table. Usually, if you put a normal rat (w/o anaesthesia) on its back, it will flip over immediately. Secondly, we use the toothed forceps to clip the feet of the rat. Because the toothed forceps is sharp at the ends, the rat will jerk its leg when it feels the pain. If it does not move its leg, it means that it is unable to feel the pain anymore. Thirdly, we use a blunt object to poke the eye of the rat. When the rat is anaesthetized, it will not blink. If it is not ready, it will blink when an object is going to hit it eye.
2. Yes, we do give more anaethesia if the rat is heavier. We calculate the amount of anaestheia to be given to the rat by its weight. Its 0.2ml of anaesthesia/100g of the body weight.
3. Actually, we do not get the cells. We obtain the corpus cavernosum tissue and culture it so that the cells of the corpus cavernosum tissue will grow. We then use the cells that we have cultured to do assays such as ELISA.
Reply to Alvin:
1.The CC of the rat and the human have the same function and therefore, we are using the rat as a model for our experiment. Both the corpus cavernosum smooth muscle of the rat and the human contains irregular spaces for blood to flow through. When the rat or human is sexually aroused, nitric oxide would be released and this would cause smooth muscle relaxation. As the smooth muscle relaxes, more blood is able to flow into the corpus cavernosum, causing it to be engorged with blood. When the CC is engorged with blood, it will cause the penis to increase in length and its regidity, thus causing penile erection. The difference is in the structure of the of the corpus cavernosum. In humans, the CC extends from the bulb of the penis and covers the penis, thus extending out of the body. For rats, the CC is only found internally. It does not extend out to cover the penis of the rat but only at the bulb of the penis found inside the body.
2. We throw away the muscles and tunica albuginea because it is of no use to us in the assay that we do. The tunica albuginea is just this white layering covering the corpus cavernosum for the CC protection, so i do not think that it is used in any work. The muscles can be used in other work such as organ baths, whereby we measure the muscle's contractility or relaxation. It all depends on what the experiment wants to find.
3. The outcome of the rat in my lab is death. The rat would be given euthanasia through the heart to relieve its suffering. Even without conducting euthanasia, the rat would still die 5-10 minutes after the removal of the bulbocavernosum because it is a very sensitive area of the rat and there are main arteries running in that area. Therefore, the removal of the bulbocavernosum to retrieve the CC within it would eventually cause death of the rat. As for mating, since the rat would die, it will not be able to mate again.
Thanks for the questions: )
Love,
Renee
TG02
0703634F
Thank you for the detailed answer! (:
ReplyDeleteLim Jia Hui (JOEY)
TG01 0703605F Group 2
hello renee~
ReplyDeleteer, how long is your cells doubling time? and what are you going to do with the cells after culture?
thanks in advance~ and cya on fri~!
yaNLing xD
Hello Yanling!
ReplyDeleteMy cells doubling time is about a week. The cells that i obtained are used for doing enzyme immunoassays.
Thanks for your question.
Love,
Renee
0703634F
TG02