title: Large scale isolation of human blood monocytes by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation for adoptive cellular immunotherapy in cancer patients.
abstract: The increasing interest in mononuclear phagocytes for adoptive cellular immunotherapy (ACI) trials in cancer patients led us to define a procedural approach to harvest reproducibly highly purified single-cell suspensions of large numbers of functional human circulating blood monocytes (Mo). A semiclosed counterflow centrifugal elutriation (CCE) system has been developed, using a new large capacity Beckman JE 5.0 rotor with one interchangeable 40 ml or 5 ml separation chamber, to purify Mo from mononuclear cell (MNC) concentrates of healthy donors and cancer patients obtained by continuous flow centrifugation leukapheresis (CFCL). This method does not require a Ficoll density gradient centrifugation step. A total of 115 leukapheresis procedures were carried out in 35 patients and in 30 healthy donors by either Cobe 2997 or Cobe Spectra, 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 procedure (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 purified Mo shows that neither morphological integrity nor physiological activity was compromised by this two-step isolation procedure, which additionally provides highly purified human Mo suspensions, in a quantity suitable for ACl of cancer patients.
authors.affiliation: Département d'Onco-Hématologie, Hôpitaux Universitaires de Strasbourg, France.
authors.forename: A
authors.initials: A
authors.lastname: Faradji
authors.forename: A
authors.initials: A
authors.lastname: Bohbot
authors.forename: M
authors.initials: M
authors.lastname: Schmitt-Goguel
authors.forename: J C
authors.initials: JC
authors.lastname: Siffert
authors.forename: S
authors.initials: S
authors.lastname: Dumont
authors.forename: M L
authors.initials: ML
authors.lastname: Wiesel
authors.forename: Y
authors.initials: Y
authors.lastname: Piemont
authors.forename: A
authors.initials: A
authors.lastname: Eischen
authors.forename: J P
authors.initials: JP
authors.lastname: Bergerat
authors.forename: J
authors.initials: J
authors.lastname: Bartholeyns
mesh_terms: D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002498:Centrifugation / Q000379:methods; D006801:Humans; D007111:Immunity, Cellular; D007116:Immunization, Passive; D016219:Immunotherapy, Adoptive / Q000379:methods*; D007937:Leukapheresis / Q000379:methods*; D009000:Monocytes / Q000166:cytology*; D009369:Neoplasms / Q000628:therapy