Good manufacturing practice-compliant validation and preparation of BM cells for the therapy of acute myocardial infarction

BACKGROUND: Intracoronary application of BM-derived cells for the treatment of acute myocardial infarction (AMI) is currently being studied intensively. Simultaneously, strict legal requirements surround the production of cells for clinical studies. Thus good manufacturing practice (GMP)-compliant collection and preparation of BM for patients with AMI was established by the Cytonet group. METHODS: As well as fulfillment of standard GMP requirements, including a manufacturing license, validation of the preparation process and the final product was performed. Whole blood (n=6) and BM (n=3) validation samples were processed under GMP conditions by gelafundin or hydroxyethylstarch sedimentation in order to reduce erythrocytes/platelets and volume and to achieve specifications defined in advance. Special attention was paid to the free potassium (<6 mmol/L), some rheologically relevant cellular characteristics (hematocrit <0.45, platelets <450 x 10(6)/mL) and the sterility of the final product. RESULTS: The data were reviewed and GMP compliance was confirmed by the German authorities (Paul-Ehrlich Institute). Forty-five BM cell preparations for clinical use were carried out following the validated methodology and standards. Additionally three selections of CD34+ BM cells for infusion were performed. All specification limits were met. Discussion In conclusion, preparation of BM cells for intracoronary application is feasible under GMP conditions. As the results of sterility testing may not be available at the time of intracoronary application, the highest possible standards to avoid bacterial and other contaminations have to be applied. The increased expense of the GMP-compliant process can be justified by higher safety for patients and better control of the final product.

Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.