ABSTRACT
Human cord blood provides a convenient alternative to bone marrow as a rich source of hemopoietic progenitor cells. This study reports a simple means for enriching a cord blood progenitor cell population by accessory cell depletion. Two methods of monocyte depletion were tested. A Cytodex 3 microcarrier system using collagen coated dextran beads was compared to the more commonly used method of plastic plate adhesion. The method of plastic plate adhesion gave a significantly higher cell recovery. T cell depletion using a recently characterized rat monoclonal antibody which fixes human complement was also investigated. A combined method of monocyte depletion by plate adhesion and T cell depletion resulted in the removal of > 96% of monocytes and > 98% of T cells. This led to a significant enrichment of myeloid (CFU-GM) and erythroid (BFU-E) colony growth. Such enriched progenitor cell populations provide a useful starting population for any study on hemopoiesis.
Subject(s)
Fetal Blood/cytology , Hematology/methods , Stem Cells , Cell Separation , Erythroid Precursor Cells , Humans , Monocytes , T-LymphocytesABSTRACT
A recently developed enzyme-linked immunosorbent assay (EIAZ, ELISA) using two murine monoclonal anti-erythropoietin antibodies was compared with a radioimmunoassay (RIA) and a commercial in-vitro bioassay, EPOS, for measuring serum erythropoietin (Epo) in humans. Specificity and validity for Epo-EIA and the other two assays were examined. The serum Epo in normal subjects was 18 +/- 12 mU/ml (mean +/- SD, n = 80) for EIA compared with 22.5 +/- 18.5 mU/ml (n = 20) for RIA and 136 +/- 132 mU/ml (n = 14) for the bioassay. The serum Epo concentrations in normals and patients were highly comparable between EIA and RIA for Epo (P less than 0.01, r = 0.95). Epo concentrations by the EIA for normal female and male subjects were 20.5 +/- 13 and 16.5 +/- 10 mU/ml, respectively. Epo levels in patients with secondary polycythaemia or autoimmune haemolytic anaemia were significantly higher than normal subjects by the three methods. Epo levels in patients with chronic renal failure were within the normal range. By the EPOS bioassay, the Epo concentrations of normals and patients with renal failure were significantly higher than expected (136 +/- 132 and 447 +/- 273, respectively). Due to its inherent design, the EPOS bioassay possibly measures bone marrow proliferative activity in response to other serum growth regulators besides erythropoietin and was found to be unsuitable for clinical assessment of Epo. We concluded that the new EIA and RIA were similarly sensitive, reliable and accurate for measurement of serum Epo. The EIA method has the advantage of being less time consuming, more convenient and avoids the use of a radioisotope.
Subject(s)
Anemia, Hemolytic, Autoimmune/blood , Erythropoietin/blood , Kidney Failure, Chronic/blood , Polycythemia/blood , Adolescent , Adult , Biological Assay/methods , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Middle Aged , Radioimmunoassay/methods , Recombinant Proteins , Reference Standards , Sensitivity and SpecificityABSTRACT
Thirteen different combinations of serum-free media were tested to assess their suitability to replace serum containing medium for in vitro culture of human hemopoietic progenitors (CFU-GM and BFU-E). Bone marrow samples from patients with and without hematological diseases were tested. All tested media supported the growth of CFU-GM and BFU-E colonies, however our results have shown that the cloning efficiency of all commercially available serum-free media tested was lower (mean 18% and 12% of controls for CFU-GM and BFU-E respectively) and the colony size was smaller than those in serum-containing medium. In the serum-free cultures, there was no linear relationship between the colony numbers and cell concentration plated. Depletion of T-lymphocytes and monocytes did not improve the cloning efficiency of the serum-free medium culture. Furthermore, the addition of high concentration of insulin, transferrin and other supplements to the serum-free media did not improve the cloning efficiency. These results have indicated that the currently available commercial serum-free media do not provide optimal requirements for hemopoietic progenitor cell cultures and that other factors contained in serum are essential for their optimal growth.
