We used enzymatic digestion method (collagenase 4 & trypsin ), but the cell number was less, around 2 X 105. Since flow cytometric analysis that we are doing need more events, there is no other way other than increasing the number of cells. Is there any better way to do the digestion? I found that hyaluronidase can improve the cell number if used for digestion. Can anyone suggest an alternative?
Dear Arun!
You look at this article, please:
A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells
https://www.nature.com/articles/s41598-019-49287-7
Preparation of a single cell suspension from human hepatic tissue
Surgically removed fresh normal liver tissues were processed and collected in an ice box at 4 °C under aseptic conditions. The tissues were quickly transported to the cell culture chamber. The specimen was placed in a Petri dish and washed 5 times with a sterile PBS solution to remove residual blood from the tissue surface (Fig. 2a,b). The connective tissues were trimmed away as much as possible, and the tissues were cut into blocks approximately 1 cm in diameter (Fig. 2c,d). The bile ducts, and blood vessels were trimmed away as much as possible using ophthalmic scissors, and the tissue were blocks were further chopped into small approximately 1-mm pieces and placed into a glass Petri dish (Fig. 2e). The premixed working solution (100 μl of penicillin-streptomycin, 250 μl of Enzyme H, 150 μl of Enzyme R, and 20 μl of Enzyme A) was added to 5 ml of DMEM and mixed well to make the mixed-enzyme solution (required for 2–4 g of tissues) (Fig. 2f). To prepare the single cell suspension of human liver tissue, the mixed-enzyme solution was prewarmed in a 37 °C water bath for 10 min before adding it to the tissues. The enzymatic treatment was carried out in the GentleMACSTM tissue processor at 37 °C for 15 minutes with constant shaking using the h_tumor_01.01 program (Fig. 2g,h).
Hello Arun
You can try a two-step process of collagenase perfusion. The steps are as follows:
Step 1. Perfuse the liver with medium containing calcium chelator (like EDTA). The calcium chelator will remove the calcium ions from the epithelial cells and as a result the cell-cell adhesion will be lost.
Step 2. Introduce collagenase solution into the liver lobes which will disrupt the extracellular matrix. Then separate the liver from the animal (I suppose, as you have not mentioned) and pass it through a 40 micron strainer by macerating it gently. The filtrate will give you single cells. Carry out this step in situ.
All the Best.
Alexandr Chernov We don't have MACS tissue processor. Could you suggest any method alternative to it?
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