The liver is the biggest organ in mammals. It plays very important role in metabolism and detoxification. Although the liver has a remarkable regeneration capacities in vitro, it is still a very big challenge for a long term to culture hepatocyte in-vitro.
Here, we establish the hepatocytes organoids from mature hepatocytes. It keeps main features of hepatocytes in morphology and function. In this video, we will introduce how to culture and passage these organoids and how to perform the genetic modification of these organoids in details.
First, liver perfusion and digestion. Prepare all the regions and the instruments and make them sterile. Wash the mouse and open the epidermal and the skin, and then try to find the pouch oven of the liver.
Inject the needles into it, and then capture this. Do the perfusion at the speed of five milliliters per minute, until the liver becomes pale. Change them into the perfusion buffer, place carefully, watch the liver, until it becomes soft.
Usually it takes three to five minutes, Then cut the liver and put them into the plates, and cut them into small pieces with the scissors. Pep it up and down frequently. Usually, it takes 10 to 15 minutes to make them into the single cell.
And then pep it up and down with 10 to 15 minutes and feed them through the fitter. Make them into the single cells. Collect all the cells in the 50 milliliters tube, and centrifuge them, with the speed and then collect all the per plates.
Try to keep them always in the cold ice. Make the single cell suspensions and count them with the cell counter. Usually the hepatocytes if happy, it has the lively fluorescent.
The second part, we try to sit the cells and do the organoid culture. We collect all the cells suspensions, and count the cells with a certain concentro-fusion And then we mix the.Metrogels. and the code medium.
Usually we take, with the medium and the metrogels, at the ratio of 1 to 2 or 1 to 3. After carefully mixing them, we sit them onto the gradient plates. We usually add 100 microliters at the 24 plate well.
Put them into the incubator at the temperature of 37 degrees. After 20 minutes, we put them reverse forward with the bottom at the bottom and then at the medium. With two or three days countering, we usually see the organoids come out.
This is the typical image of our organoids. We usually need single cells from organoids if we want to do the passage or do some genetic modification method We collect all the organoids from the countering plates with the coat wash buffer. We drop the supernatant, then at the coat wash buffer into the plate, mix them, so pep it up and down.
And then we usually pre-wash all the tips with the wash tapes buffer. To avoid all the organoids tip onto the tips. And then with the, a new batch on ice or without it, we collect all the organoids by centro-fusion.
We treat them, there's a conning radients to remove all the metrogels. After 20 to 30 minutes of incubation on ice all the metrogels are removed. We collect all the clean organoids and add trypsin to make them into single cells.
After trypsin is added, we treat the organoids into the incubator. It takes 5 to 10 minutes for liver organoids to become single cells. We add more wash buffers to stop the triplatin.
And then pep it up and down towards them. For option process, you can wash them one more time to remove all the trypsin. After wash by centrofusion, we can count them under the cell counter.
Then we will show how to do transaction or transfection of these hepatocytes organoids. First, label the tubes of what you will do, and then we'll do the eyesight transfection according to the manufacturer's instructions. You need, the regent of lipoyl fact taming INI MaX.
We add the. control and the specific gene siRNA, and incubate them with the optimum, according to the manufacturer's instructions. And then according to the cell concentration we add the RAN max and mix them thoroughly.
Then we also add the RAN max with the optimum separately and put them on ice. After 5 minutes, we pep up separate RAN max mixture with different siRNAs, Specific gene RANi and II control separately and mix them thoroughly. Put them on ice again, and then we collect all the organoids, slow centrofusion, drop all the supernatant, we add the RNAi max, the siRNA mixture into this cell plate and pep it up and down thoroughly.
Briefly, the cells and the label RNAi max and siRNA mixture were gentrification and incubate for 4 to 5 hours at 37 degrees. Final part, these hepatocytes organoids could also be infected by the Lenti virus. The similar message was performed as siRNA transfection.
The control and the siRNA virus tubes was well prepared, And the optimum was added according to the manufacturer's instructions. Then with the infection regions like poly green and then we add different Lenti virus, according to the manufacturer's instructions constitution. Combine the single cell suspensions in organoids culture and various particles in the 1.5 milliliters eppendorf tubes, Add, well, mix them Plate this mixture, and concentrate them for 1 hour at 32 degrees.
The concentrate fusion will be performed at 500 G.And after 4 to 5 hours incubation at 37 degrees the cell suspension will then be collected and seated in metrogel. And put them into the incubator again. The organ on the formation efficiency or other functional analysis could be performed 7 to 10 days later.
Here is our representative results. In figure 1A, you could see our ALB-Cre Rosa 26-GFP or RFP hepatocytes organoid from mouse. In figure 1B you could see the Ki67 positive signal in staining, which has shown that our hepatocytes organoids are proliferating.
In figure 2A and 2B here is a representative gene expression of our interfering expression of our genes. After genetic manipulation in hepatocytes organoids, the interfering effective is very efficient. Here is figure 2C, you could see after genetic manipulation with carsonite technology in our murine organoids.
Conclusion, our results showed the hepatocytes organoids could be expanded in vitro and genetically manipulated. In summary, in this video we show how to do the liver perfusion and digestion to get the hepatocytes. How to capture the hepatocytes and passage the hepatocytes organoids.
Also, we showed how to do the siRNA and vector transfection or Lenti virus infection to do the modification of the genetic of these organoids. Also, we have two shortages in our organoids. First, we didn't use per core or fur core to pure the hepatocytes.
And second I think more growth factors and signaling should be tried to improve the organoids growing time.
