The overall goal of the following experiment is to obtain multiple macrophage cultures from mixed primary cultures of rat liver cells without the need for complex equipment or skills. This is achieved first through dissociation of adult rat liver cells by collagenase perfusion, followed by isolation of the parenchymal hepatocyte cell fraction. Next, the cells are incubated in T 75, tissue culture flasks with growth medium to establish mixed primary cultures of liver cells.
Then after 10 to 12 days of culture, the culture flasks are shaken to facilitate transfer of the macrophages to plastic dishes from which they're then harvested by selective adhesion after a short incubation period. Finally, more than 10 of the six cells can be harvested repeatedly from the same T 75 tissue culture flasks at two to three day intervals for more than two weeks. Isolation methods for liver macrophages have been well described.
However, most of the previous methods require sophisticated equipment such as a centrifugal illustrator and refined technical skills. Here we present a simple and novel methods to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult wrapped liver cells that requires no special equipment or advanced laboratory skills. Animal dissection, perfusion of the liver and preparation of cytes will be demonstrated by doctors Norco, Yamanaka, and Miko yoshioka at the National Institute of Animal Health.
Then isolation and purification of liver macrophages from cultural risks will be shown by Dr.Taketo Takeno at the National Institute of Agro Biological Sciences. So let's get started. To prepare for profusion of the rat liver begin by prewarm one bottle of washing solution and one bottle of collagenase solution.
Then after confirming sedation by skin pinch place an anesthetized adult male rat in a stainless steel pan and spray 70%ethanol onto its abdomen and thorax. Next, make a small cut in the middle of the abdomen with dissecting scissors and peel back the skin. Then open the abdomen with the scissors and confirm the location of the portal vein.
Next, pass the surgical thread under the portal vein and loosely tighten the thread. Then clamp the distal part of the inferior vanoc cava and mesenteric vein with a mosquito clamp to prevent blood reflux into the liver. Open the thoracic cavity by cutting the diaphragm with scissors and expose the heart after making a small incision in the portal vein with ophthalmic scissors, insert a two millimeter plastic catheter into the vein and tighten the thread firmly around the catheter.
Load a perfusion system with the prewarm washing solution and then connect it to the catheter. Begin perfusion in C two at a rate of 10 milliliters per minute. At the same time, make a cut in the right atrium wall with dissecting scissors.
Continue the profusion for 10 minutes, then switch the profusion solution to the collagenase solution and perfuse for 10 to 20 minutes. At a rate of 10 milliliters per minute, fill a sterile beaker with 25 milliliters of collagenase solution. Then remove and carefully place the perfuse liver into the beaker.
Mince the liver into small pieces with scissors. Add 75 milliliters of cold MEM into the resultant cell suspension. After gentle pipetting, filter the suspension through a 100 micrometer cell strainer.
To remove the connective tissue in the undigested tissue fragments, collect the filtrate into 50 milliliter conical tubes. Transfer the filtrate into 50 milliliter conical tubes and wash the cells four times by first spinning down the cells at 50 Gs for one minute at four degrees Celsius. With the break off, carefully discard the supernatant and then resuspend the pellet in cold MEM and spin the cells down again after the last wash.
Resuspend the liver cells in growth medium. Now seed the cells in five to 10 T 75 tissue culture flasks at a density of 6.7 times 10 to the fourth cells per square centimeter. Place the culture flasks in an incubator and replace the growth medium every two to three days.
After one day of culture, parenchymal hepatocytes spread on the flask surface and show typical polygonal copal stonelike morphology with one or two round nuclei. Within a few days of culture, parenchymal hepatocytes lose their epithelial cell morphology and transform into more flattened fibroblastic cells Around day six, phase contrast, bright round macrophage like cells start to proliferate on the fibroblastic cell sheet. The growth of the macrophage like cells reaches maximum levels around day 12.
The number of macrophage like cells declines around day 19 when the fibroblastic cell sheet starts to degenerate. Thus around seven to 10 days of culture, we suspend the macrophages that have proliferated on the cell sheet into the culture medium by reciprocal shaking of the culture flasks for 30 minutes after the macrophages have been resuspended. Play each of the 100 millimeter non tissue culture grade plastic dishes with the medium from two T 75 flasks.
Refill the culture flasks with growth medium and return them to the incubator. Incubate the plastic dishes for 30 minutes after the incubation period, wash the dishes three times by aspirating the medium and gently rinsing the plastic dish with PBS as seen here as early as 10 minutes after plating macrophage like cells attached to the dish surface while other contaminating fibroblastic cells remain suspended. After the PBS rinse, a highly purified macrophage population is obtained.
As seen in this figure, the cells gain typical macrophage morphology after 40 minutes of culture and mitotic cells are frequently observed as indicated by the arrows. To collect this new highly purified macrophage population at one milliliter of trip LE Express solution to the washed cells and place the dishes back in the incubator for an additional 10 minutes. After this incubation period, add nine milliliters of growth medium and then gently scrape off the attached macrophages with the cell scraper and transfer into a 15 milliliter conical tube centrifuge and discard the supernatant.
Add one milliliter of the growth medium, dissociate the resuspended cell clumps into single cells by vigorous pipetting, enumerate the cell number by hemo cytometer as shown in this graph, more than 10 of the six cells can be harvested from a T 75 culture flask repeatedly at two to three day intervals for more than two weeks, enabling a total cell yield of 10 to the seven per T 75 Culture flask. As shown in these images, the isolated cells are immuno stain with monoclonal antibodies against rat macrophage antigens such as CD 68 or ED one and CD 1 72 A or OX 41. These cells possessed functional properties of macrophages such as active phagocytosis of FITC labeled microbeads.
In this video, we presented a simple and efficient method to obtain macrophages from mixed primary cultures of adult red liver cells. Our procedure does not require complex equipment or skills, but provides sufficient numbers of pure macrophages that can be repeatedly harvested from the same culture of brass. This method can be applied to other mammalian species.
Thank you for watching the video and good luck in your future experiments.Cheers.
