Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells

Abstract

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/ progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34 + HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34 + cells are expanded 30-to 65-fold or 400-to 800-fold, yielding a purity of »80% and »60% CD34 + cells, respectively. This system was adapted to a 96well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 10 3 cells per well to determine drug IC 50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC 50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Ex Vivo Chemosensitivity Profiling of Acute Myeloid Leukemia and Its Correlation With Clinical Response and Outcome to Chemotherapy

“…The in-vitro single-cell culture methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ATP assay cannot distinguish between tumor cells and non-tumor cells, or simulate the in-vivo microenvironment (8)(9)(10). Nowadays, high-throughput ex-vivo drug sensitivity screening test is used to detect pharmacological activity in the treatment of AML (11)(12)(13)(14)(15)(16)(17)(18). Herein, we tested the prediction value of the PharmaFlow platform using bone marrow samples of 104 AML patients.…”

Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs

“…To this aim, we applied a novel flow cytometer gating strategy to prospectively identify the most primitive HSPC subpopulation as CD45 + , Lin − , CD34 high , CD90 + , CD45RA − cells ( Figure S3 A). 37 , 38 , 39 , 40 , 41 , 42 Human HSPCs proliferated in culture in the presence of cytokines, reaching a significantly higher number of cells (p = 0.0079) when co-cultured with MSCs ( Figure S2 B). Phenotypic analysis resulted in a significantly higher number of HSPCs with a primitive phenotype (p = 0.02) ( Figures S2 C and S2D) characterized by an increased clonogenic capacity (p = 0.02) ( Figure S2 E) when cultured in the presence of MSCs compared with controls.…”

Standardized assays to monitor drug sensitivity in hematologic cancers

“…Some of these cytokines include FLT3L, SCF, TPO, G-CSF, GM-CSF, IL-3, IL-6, and IL-1B [ 54 , 55 ]. A common approach to improve cell viability and to stimulate blast proliferation is to use serum-free medium, such as StemSpan SFEM II, supplemented with the selected cytokines [ 56 ]. In addition, conditioned medium derived from HS-5 human stromal fibroblast cell line has been employed to enhance blast survival [ 57 ].…”