Abstract:    For cells which need to be digested and cultured for passage, each digestion significantly tests whether the cell can survive and is in a good state. Many researchers find their cultured cells grow well at the very beginning of generations. After passaging for several generations, the cells are in a bad state. It's quite sure that there is something wrong with the digestive process. Cells are greatly hurt in each digestion and grows worse.
Keywords:  Cell Culture, Cell Digestion, Cell Subculture

1. Introduction

During digestion, all cells are digested by added pancreatin. However, we ignore a problem: The cell growth state is different. The adherent effect of each cell also varies. Some cells adhere firmly and others adhere loosely. It's unfair for cells to be digested for the same time. Then the digestive method is improved by using a four-step digestion(Take cells hard to be digested for example).

2. Four-step Digestion

Step 1. First, don't add pancreatin. Discard the old medium and add a little new medium for washing 1-2 times.(This is the prelude and the first step, aiming to wash floating dead cells)

Step 2. Readd a little new medium by direct pipetting. Cells which adhere loosely are removed. Then, rewash with the medium. The mixed suspension in the first two steps is transmitted into new bottle.(The cell sensitivity for pancreatin can be observed for the growth state by comparing with cells digested by pancreatin)

Step 3. Absorb the residual liquid in the bottle completely and wash by adding about 0.3ml pancreatin. Then absorb and remove the washing solution. Readd about 1ml pancreatin for digestion and observe under a microscope during digestion. When intercellular space is obvious, pancreatin shall be absorbed and discarded immediately. Then, add new medium for pipetting 2-3 times and absorb suspension into test tube or new bottle for temporary storage. Wash with 2ml new medium and mix with previous solution. (Or subculture the cells in new bottle and compare the difference)

Step 4. Then add new pancreatin into the bottle to continue digest rest cells firmly adhering. Still observe under a microscope. When intercellular spaces of rest cells are evident and cells are independent, pancreatin should be absorbed. Then add new medium for pipetting. Suspension shall be transmitted into new bottle for culturing(Adjust according to the experimental process).

For cells which have great difficulties in digestion or are specially sensitive for pancreatin, the fifth or sixth step can be added. Lot-to-lot digestion is suggested for better cellular state and more beautiful morphology. Then, the pancreatin's damage to cells can be deceased to the maximum. Cells can be kept in the best state.

Of course, if cells are easy to be digested like RAW264.7, the second step is just sufficient. The third and fourth step can be ignored. The details are mastered during your experience. Upon receipt of a new cell, you are suggested to respectively culture cell digested in each step for comparison. Cells for pancreatin's requirements can be discovered for the subsequent experience.

3. Conclusion

Besides the avoidance of pancreatin's damage to cells, the four-step digestion plays a more important role in maximally pipetting cells into single cellular state. Confluence and continuity between cells are prevented.

Because cells interrelate with each other during the growth, most cells grow confluent. Especially for adherent cells, after single cell adheres, they will grow confluent. If confluent cells are in a cluster, adhension of cells shall be avoided after subculture. Otherwise, cells in a cluster are dead. Thus, cells shall be digested into single independent state during digestion. This is a great test for experimenters.

Once cells fall into suspension, they are hard to be pipette for single one without the stress point. It's very hard to find the stress point to separate the cell. Cells should be pipette before falling. The stress point is the place where cell adhesion happens. The digestive degree shall be strictly controlled. Cells should be digested for easy pipetting instead of over-pipetting and should individually fall instead of confluent falling. Adding pancreatin in batches ensures adherent cells in different growth state can be separated with good stress point. This is the fatal point and important for some cells like Caco-2 cell.

There is another important point for subculture. Cells shouldn't be digested till they overgrow. When cells grow by 70%, the subculture should be performed. Once the folded growth is found during cells growth, digestion and subculture should be conducted immediately.