Erwin Strasser

Different preparation methods to obtain platelet components as a source of growth factors for local application

BACKGROUND: Autologous platelet components were recently used as part of tissue‐engineering strategies in oral and maxillofacial surgery. Various preparation methods were investigated to define standardized blood bank components and to collect data on the growth factor content of human platelets before and after storage.

Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories™

Dendritic cells (DC) are increasingly used for the immunotherapy of cancer. Both the induction of tumor-specific T cells and some clinical regressions have been observed in early phase I/II trials by using either DC isolated from blood, DC generated from CD34+ precursors ex vivo, and most frequently, by employing monocyte-derived DC. As DC vaccination is now awaiting phase II/III trials with larger patient collectives, it becomes increasingly important to overcome prior limitations such as the repetitive, labor-intensive generation of DC in a large number of open culture vessels. We describe here as a result of several years of optimization, in detail, a procedure that uses the so-called Nunc cell factories to process a whole apheresis product, labor- and cost-effectively in a quasi-closed system to reproducibly generate (by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha +PGE(2)) large numbers (8.32+/-3.8% of input peripheral blood mononuclear cells (PBMC)) of mature (>85% CD83+), monocyte-derived DC that can be successfully cryopreserved. Our report is based on the processing of >100 aphereses including 52 unselected aphereses in advanced melanoma patients. This allows us also to suggest meaningful quality and validation criteria. The DC generation method appears particularly promising as respective DC vaccination proved to be immunogenic in cancer patients and cell factories can readily be converted to a fully closed system by using appropriate valves, tubings, and bags.

Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates

Background and Objectives  Autologous platelet concentrate (PC) is applied locally to improve wound healing and tissue repair. Previous measurements of the growth factor content of platelets have given conflicting results. To date, there is no information on the influence of different preanalytical sample‐preparation methods on the detectable amount of growth factors.

Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection

Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is the current lack of a simple functionally “closed” system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized the use of the Elutra counterflow elutriation system to enrich monocytic DC precursors by (1) developing an algorithm to avoid red blood cell debulking and associated monocyte loss before elutriation, and (2) by elutriation directly in culture medium rather than phosphate-buffered saline. Upon elutriation the bags containing the collected monocytes are simply transferred into the incubator to generate DC progeny as the final “open” washing step is no longer required. Elutriation resulted in significantly more (≥2-fold) and purer DC than the standard gradient centrifugation/adherence-based monocyte enrichment, whereas morphology, maturation markers, viability, migratory capacity, and T cell stimulatory capacity were identical. Subsequently, we compared RNA transfection, as this is an increasingly used approach to load DC with antigen. Elutra-derived and adherence-derived DC could be electroporated with similar, high efficiency (on average >85% green fluorescence protein positive), and appeared also equal in antigen expression kinetics. Both Elutra-derived and adherence-derived DC, when loaded with the MelanA peptide or electroporated with MelanA RNA, showed a high T cell stimulation capacity, that is, priming of MelanA-specific CD8+ T cells. Our optimized Elutra-based procedure is straightforward, clearly superior to the standard gradient centrifugation/plastic adherence protocol, and now allows the generation of large numbers of peptide-loaded or RNA-transfected DC in a functionally closed system.

Hyperconcentrated platelets stored in additive solution: aspects on productivity and in vitro quality

Background and Objectives  New platelet (PLT) additive solutions (PASs) allow a plasma carryover of < 30% in PLT concentrates. This implicates the need to collect apheresis PLT concentrates at very high PLT concentrations: so‐called dry PLTs (DPs). We used the TRIMA, with software version 4 (TRIMA V4), to collect such DPs and investigated the in vitro quality of these PLTs when stored in the new modified PAS‐III (PAS‐IIIM).

Contribution of MINCLE–SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk–Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF–driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA–mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK–CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.

Cord blood processing with an automated and functionally closed system

BACKGROUND: Umbilical cord blood processing with standard centrifugation techniques is performed in open systems and results in varying cell and volume recoveries.

Washing platelets with new additive solutions: aspects on the in vitro quality after 48 hours of storage

BACKGROUND:  Rare clinical conditions cause the need for washed platelet (PLT) concentrates (PCs). Saline‐washed PCs can only be stored shortly, however, owing to lack of substrates for PLT metabolism. New PLT additive solutions (PASs) contain such substrates and might be used alternatively. The in vitro quality of apheresis PCs washed with Composol‐PS or modified PAS‐III (PAS‐IIIM) stored up to 48 hours after wash was compared.

First comparison of productivity and citrate donor load between the Trima® version 4 (dual‐stage filler) and the Trima Accel® (single‐stage filler) in the same donors

Background and Objectives  Aside from new software the blood cell separator TRIMA (GambroBCT) also received a newly designed separation chamber offering a novel single stage separation technology, called Trima Accel. We evaluated this new system focusing on productivity and donor comfort by comparing it to the previous version (Trima version 4) in collecting single‐donor platelet concentrates (SD‐PCs) and plasma.

The in vitro quality of washed, prestorage leucocyte-depleted red blood cell concentrates.

Background and Objectives  No data are currently available on the quality of washed prestorage leucocyte‐depleted red blood cell concentrates (RCCs).