Analysis for Recovery and Loss of Mononuclear Cells and Colony‐Forming Units Granulocyte‐Macrophage during ex vivo Processing of Autologous Bone Marrow

Abstract

During ex vivo processing of autologous bone marrow (BM) substantial loss of stem and progenitor cells should be avoided to achieve rapid and sustained hematopoietic reconstitution after high-dose radio-/chemotherapy. We processed 25 autologous BM grafts with the Fresenius AS104 cell separator for cryopreservation and we determined recoveries for mononuclear cells (MNC) and colonyforming units granulocyte-macrophage (CFU-GM) in the BM concentrates. To identify cell loss in BM fractions not cryopreserved, we investigated the MNC and CFU-GM content of BM fat and BM blood. MNC and CFU-GM recovery yielded a mean ( +/- SEM) of 42 +/- 12 and 54 +/- 20% in the BM concentrate. BM fat showed a mean loss of 7 +/- 5% for MNC and 4 +/- 3% for CFU-GM, BM blood 30 +/- 12% for MNC and 13 +/- 13% for CFU-GM, respectively. CFU-GM recovery was significantly higher in the BM concentrate of patients with hematologic malignancy (HM) compared with patients suffering from germ cell cancer (GCC): 66 +/- 21 vs. 43 +/- 12% (p < 0.02). Seventeen patients (7 GCC, 10 HM) underwent high-dose chemotherapy or radio-/chemotherapy and were autografted with 0.8 +/- 0.2 x 10(8) MNC/kg and 3.7 +/- 2.0 x 10(4) CFU-GM/kg. All patients achieved engraftment with neutrophils > 0.5 x 10(9)/l at a mean of 14 +/- 6 days. We conclude that: (1) ex vivo processing of autologous BM with a mean of recovery of 42% for MNC and 54% for CFU-GM in the BM concentrate can result in a cell population capable of sustained hematopoietic reconstitution, (2) CFU-GM recovery is significantly higher in patients with HM than in patients with GCC and (3) 37% MNC and 17% CFU-GM represent in fact cell losses recovered from BM fractions not cryopreserved (BM fat, BM blood). Furthermore, it is likely that MNC and CFU-GM not recovered from BM concentrate, BM fat and BM blood are cell losses related to the cell separator.

In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects

“…The average density of mononuclear cells in human bone marrow is (123±74) × 10 8 cells/ml. 34 Chondrocytes account for only 1–2% of cartilage volume, 35 and MSC therapy is suggested at a density of (5.8±2.3) × 10 6 cells/ml. 36 Thus, BMMNCs are mathematically sufficient for cartilage repair.…”