AS CULTURED CELLS GROW AND DIVIDE, THEY USE UP NUTRIENTS AND EVENTUALLY, CELLS WILL MULTIPLY
TO A POINT THAT THEY RUN OUT OF SPACE IN THE CULTURE VESSEL.
SUBCULTURING IS THE PROCESS OF DILUTING AND TRANSFERRING THE CELLS INTO MORE VESSELS WITH
FRESH CULTURE MEDIUM SO WE CAN PRODUCE A NEW CULTURE AND THE CELLS CAN CONTINUE GROWING.
IN THIS EXPERIMENT WE’LL DEMONSTRATE THE STEPS FOR PROPAGATING A CULTURE BY TRANSFERRING
NIH3T3 CELLS FROM A PREVIOUS CULTURE TO A NEW CULTURE VESSEL.
TO BEGIN, REMOVE THE CULTURED CELLS FROM THE INCUBATOR.
VISUALLY INSPECT THE CELLS FOR A CHANGE IN PH, WHICH CAN BE DETECTED BY A DISCOLORATION
OF THE INDICATOR PHENOL RED.
EXAMINE THE CELL CULTURE UNDER AN INVERTED MICROSCOPE.
LOOK FOR ANY SIGNS OF CONTAMINATION OR UNHEALTHY CELLS.
THESE CELLS LOOK HEALTHY.
NEXT, CALCULATE THE PERCENT CONFLUENCY.
CONFLUENCY IS THE PERCENTAGE OF THE VESSEL COVERED BY CELLS.
OUR CELLS ARE AT ABOUT 90-PERCENT CONFLUENCY.
UNDER THE VERTICAL FLOWHOOD, REMOVE AND DISCARD THE CULTURE MEDIUM INTO A WASTE BEAKER THAT
CONTAINS APPROXIMATELY 100 ML OF BLEACH.
THE BLEACH IS USED TO KILL ANY CELLS OR MICROBES THAT ARE LEFT OVER.
BRIEFLY RINSE THE CELL LAYER WITH 5 MILLILITERS OF STERILE PBS TO REMOVE ALL TRACES OF THE
SERUM, WHICH CONTAINS A TRYPSIN INHIBITOR.
ADD 3 ML OF TRYPSIN SOLUTION TO THE T-75 FLASK.
MAKE SURE THE TRYPSIN HAS REACHED THE ENTIRE SURFACE AREA OF THE SUBSTRATE.
IT’S IMPORTANT TO LIMIT THE CELLS’ EXPOSURE TO TRYPSIN TO THE LEAST AMOUNT OF TIME POSSIBLE…
JUST LONG ENOUGH FOR THE CELLS TO DETACH.
THE TIME VARIES DEPENDING ON THE CELL LINES.
WE’LL INCUBATE THESE CELLS FOR 2 MINUTES.
THIS WILL HELP QUICKEN THE PROCESS OF DETACHING THE CELLS FROM THE SUBSTRATE.
REMOVE THE FLASK FROM THE INCUBATOR AND USE AN INVERTED MICROSCOPE TO CHECK FOR DISPERSION.
THE CELLS IN OUR CULTURE ARE PROPERLY DISPERSED.
AS YOU CAN SEE, THE CELLS ARE DETACHED AND FLOATING.
BACK UNDER THE HOOD, ADD 7 ML OF COMPLETE CULTURE MEDIUM WITH 10% SERUM TO THE TRYPSINIZED
CELLS.
AND RESUSPEND THE CELLS BY PIPETTING UP AND DOWN SEVERAL TIMES.
TRANSFER THE CELL SUSPENSION TO A 50-ML CONICAL TUBE.
NEXT, PLACE THE TUBE INTO THE CENTRIFUGE.
WE NEED TO ADD A SECOND TUBE FOR BALANCE.
IT SHOULD HAVE THE SAME VOLUME AND BE PLACED DIRECTLY OPPOSITE OF THE TUBE THAT CONTAINS
OUR CELLS.
CENTRIFUGE THE CELLS AT 125-G FOR 5 MINUTES.
ONCE THE CELLS HAVE FINISHED SPINNING, LOOK FOR A PELLET AT THE BOTTOM OF THE TUBE.
HOLD THE TUBE STEADY BECAUSE SHAKING THE TUBE CAN DISPLACE THE PELLET.
USE A PIPETTE TO CAREFULLY REMOVE THE SUPERNATANT, WHICH IS THE LIQUID ABOVE THE PELLET.
ADD 5 ML OF FRESH PRE-WARMED CULTURE MEDIUM TO THE TUBE.
RESUSPEND YOUR CELL PELLET BY PIPETTING UP AND DOWN.
COUNT THE CELLS USING A HEMOCYTOMETER AS SEEN HERE, OR USE AN ELECTRONIC CELL COUNTER.
CLICK THE LINK ON YOUR SCREEN TO WATCH A DEMONSTRATION OF THESE STEPS.
BASED ON YOUR CELL COUNT, TRANSFER THE APPROPRIATE NUMBER OF CELLS TO A NEW CULTURE VESSEL.
BRING THE VOLUME UP TO 25 ML USING FRESH, PRE-WARMED CULTURE MEDIUM.
IT'S A GOOD IDEA TO CHECK THE CULTURE VESSEL UNDER AN INVERTED MICROSCOPE TO MAKE SURE
THE CELL TRANSFER WAS SUCCESSFUL.
WE CAN SEE CELLS, SO THIS CULTURE IS READY FOR THE INCUBATOR.
INCUBATE THE CULTURE AT 37 DEGREES CELSIUS.
NOW THE CELLS ARE ABLE TO CONTINUE GROWING AND DIVIDING.