Table 5.

Protocol currently recommended for CTC cell lines.

1 First step

(1) Blood sample volume >10 mL

(2) Negative selection for CTC enrichment (RosetteSep®, etc.) or Ficoll-Paque density gradient centrifugation

(3) Hypoxic conditions within 10 days (7 days is recommended)

(4) Low attachment plate or gel formation

(5) Growth factors (such as EGF, FGF, and B-27) and anti-apoptotic supplements (including Y-27632) with FCS

2 Option

(1) Preservation of CD45(+) cells.

(2) Normoxic conditions and attachment plates dependent on the cell type

(3) Microfluidic device or nanotechnology-based method

(4) Serum-free medium

(5) Co-culture with normal leukocytes or fibroblasts

(6) Culture medium changes under the microscopic ministering of cell clusters

(7) Exclusion criteria

(i) the use of chemotherapy and/or antibody-based therapy in the past 4 weeks

(ii) the use of radiotherapy in the past 2 weeks

3 Second step (after 7 to 14 days of the initial culture)

(1) Transfer to normoxic conditions

(2) Standard culture medium

4 Third step (after several weeks)

(1) No standard method

(2) Select specific conditions depending on each cell

(3) Careful management

5 Critical steps

(1) Medium changes at 200 μL/min or less every 72 h [64]

(2) In the case of a highly proliferative culture, an increased frequency of medium changes

(3) To prevent the excessive stimulation of normal blood cells, the use of anti-apoptotic supplements for long periods is not recommended