The invention discloses a
perfusion culture method for increasing the upper limit of
living cell density before seed transformation in a
cell culture N-1 stage, and relates to the technical field of
cell culture. The method comprises the following steps: supplementing a basic culture medium on the 3rd day of wave culture, carrying out gradient increasing
perfusion by using the basic culture medium at different
perfusion rates every day from the 5th day,
monitoring pH and DO parameters every day, enabling cells to be in a high-density and high-
motility growth state, removing the waste culture medium through a
cell interception membrane in a disposable wave perfusion bag, and finally obtaining the high-density and high-
motility cells. The cells are retained in the culture bag, and the
living cell density upper limit before seed transformation is improved. According to the method, a culture mode of supplementing materials and then performing perfusion is adopted, so that 200L and 500L reactor processes in a common culture process are omitted, a 2000L reactor can be directly connected for culture after a wave process, and the time and the cost of a
cell culture process are saved.