LARGE-SCALE ISOLATION OF HUMAN BLOOD MONOCYTES BY CONTINUOUS-FLOW CENTRIFUGATION LEUKAPHERESIS AND COUNTERFLOW CENTRIFUGATION ELUTRIATION FOR ADOPTIVE CELLULAR IMMUNOTHERAPY IN CANCER-PATIENTS

The increasing interest in mononuclear phagocytes for adoptive cellula r immunotherapy (ACI) trials in cancer patients led us to define a pro cedural approach to harvest reproducibly highly purified single-cell s uspensions of large numbers of functional human circulating blood mono cytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) sys tem has been developed, using a new large capacity Beckman JE 5.0 roto r with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and can cer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrif ugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe S pectra, with a similar efficiency in MNC apheresis. The average yield per leukapheresis procedure was 5.6 x 10(9) MNC of purity 90-100% (25- 45% Mo, 40-65% lymphocytes). The average yields per elutriation proced ure (R/O fraction) were 1.1 x 10(9) cells (purity 93% Mo) using the 5 ml separation chamber, and 1.5 x 10(9) cells (purity 91%) using the 40 ml separation chamber, with a respective recovery of 82+/-7% and 78+/ -8% Mo. In vitro analysis of the viability and function of the purifie d Mo shows that neither morphological integrity nor physiological acti vity was compromised by this two-step isolation procedure, which addit ionally provides highly purified human Mo suspensions, in a quantity s uitable for ACl of cancer patients.