To Isolate human umbilical arterial smooth MUS cells. I used the following solutions. PSS, physiologic SELINE solution FBS fatal B, serum inactivate A B, antibiotic and anti mycotic rrp, M-I-P-B-S phosphate buffer selling solution, DM A F 12 collagenase and HBSS ANGs balanced selling solution to dis solve the collagenase Type one.
In terms of manipulation of the samples, all process is done in laminar airflow cabin. The amical cart is cutting into small pieces between three and seven centimeters. Next, the pieces are put into a bigger container with PSS antibiotic, anti mycotic, and anti proteases to avoid deterioration or a loss of cell viability.
The isolation is done at the temperature of four cells degrees. The researcher is now indicating the vein and arteries that are present in the umbilical cord. Waterton jelly that surrounds the arteries is carefully removed by cutting.
We scissors to Remove double of the remaining inside the vessels. The arteries are vertically held and profused with PSS using a serene connected to a TEFL needle if necessary, this washing step can be repeated for several times. The same processor is repeated using RPMI supplemented with 10%of FPS.
Then the end of arteries are closed. Next, the arteries are profused with collagenase type one. Then the upper, upper standard arteries are also closed.
The arteries Are incubated with PBS for 15 minutes at the temperature of 37 CEL degrees with some vegetation After incubation, the ends are cut to collect the free smooth muscle cells. The arteries Are profused with demand F 12 supplemented with 5%of FPS antibiotic and anti Mycotic. The medium used to peruse the arteries is Collect to a tube.
Tube is centrifuged at the room temperature along eight minutes at to hundred 50 gs. The centrifugation can be done in different rotations depending on the type of cells. If the speed is excessive cell damage can occur.
The S natant is aspirated with the half of a past PPE connected to a vacuum pump. The pallet is reus suspended in five milliliters of D DM F 12, supplemented with 5%of FPS so that the cells can be placed on a T flask where they will grow. Collagen Was added to the bottom of the T flask in order to allow cell growth.
Collagen is used to cover the bottom of T flask because one of the great difficulties we have in this method is to be able that cells adhere so it is coated with extracellular matrix proteins. Cells Are observed with the help of the inverter, optical microscope to see before incubation. The cells are incubated at the temperature of 37 cells degrees under a OMI five atmosphere with 5%of carbon dioxide After five to 16 hours.
The non-ad cells and cellular diaries are removed with either of a pasture P PET connected to a vacuum Pump. Fresh culture medium is added to the T flask. The Cells Are incubated at a temperature of 37 CEL degrees and modified atmosphere with 5%of carbon dioxide.
Again, the cell culture medium is changed every two to three days until smooth muscle cells reach confluence. After cells detain confluence, they are Seeded into two or three cell culture flask.
