The present invention has an object to solve the problem in a method for separating nucleated cells from a cell-containing fluid using a cell separation filter. Specifically, the present invention provides a cell separation filter and a method for preparing cells using a cell separation filter, which are capable of reducing unnecessary cell contamination in a nucleated cell fraction without needs of designing non-woven fabrics to suit cell species to be recovered. The present invention also provides a cell separation filter and a method for preparing cells using the filter capable of improving the recovery yield of nucleated cells. The present invention provides a cell separation filter comprising: a container having an inlet and an outlet, non-woven fabrics laminated in the container, and a partition having an opening, the partition separating the non-woven fabrics. The present invention also provides a method for preparing nucleated cells including the steps of: introducing a cell-containing fluid through the inlet of the cell separation filter to contact the cell-containing fluid with the non-woven fabrics and recovering a nucleated cell fraction from the filter.
