Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells

Abstract

Stem cells hold great promise as a means of treating otherwise incurable, degenerative diseases due to their ability both to self-renew and differentiate. However, stem cell damage can also play a role in the disease with the formation of solid tumors and leukaemias such as chronic myeloid leukaemia (CML), a hematopoietic stem cell (HSC) disorder. Despite recent medical advances, CML remains incurable by drug therapy. Understanding the mechanisms which govern chemoresistance of individual stem cell leukaemias may therefore require analysis at the single cell level. This task is not trivial using current technologies given that isolating HSCs is difficult, expensive, and inefficient due to low cell yield from patients. In addition, hematopoietic cells are largely non-adherent and thus difficult to study over time using conventional cell culture techniques. Hence, there is a need for new microfluidic platforms that allow the functional interrogation of hundreds of non-adherent single cells in parallel. We demonstrate the ability to perform assays, normally performed on the macroscopic scale, within the microfluidic platform using minimal reagents and low numbers of primary cells. We investigated normal and CML stem cell responses to the tyrosine kinase inhibitor, dasatinib, a drug approved for the treatment of CML. Dynamic, on-chip three-color cell viability assays revealed that differences in the responses of normal and CML stem/progenitor cells to dasatinib were observed even in the early phases of exposure, during which time normal cells exhibit a significantly elevated cell death rate, as compared to both controls and CML cells. Further studies show that dasatinib does, however, markedly reduce CML stem/progenitor cell migration in situ.

Cell chip array for microfluidic proteomics enabling rapid in situ assessment of intracellular protein phosphorylation

“…In this configuration, live CD34+ stem/progenitor cells were preloaded with the intracellular mitochondrial stain, tetramethylrhodamineester ͑TMRM͒ ͑Invitrogen͒ and perfused with serum free medium containing 5 growth factors 5 and 1 nM Sytox Green to label dead cells. In a similar fashion as our previous work, 5 this illustrates that live cell dynamics information can be obtained on single cells registered in the array, viability and mitochondria location and quantities, for example. These cells were then exposed to a fixation solution containing 20% v/v Cytofix/ Cytoperm ͑Becton Dickenson͒, which resulted in the depolarization of mitochondria and dispersal of the TMRM dye out of the cells, in less than 2 min.…”

“…5,14 Briefly, SU-8 masters were used to cast polydimethyl siloxane ͑PDMS͒ ͑Dow Corning͒ devices, which were irreversibly bonded to glass cover slips using plasma oxidation. Prior to use, devices were sterilized by immersion in a sonic bath containing methanol for 5 min and were then flushed with sterile cell culture media.…”

“…[3][4][5][6] For example, sieves have been designed to minimize the influence of liquid flow on adherent MCF7 breast cancer cells 7 and create high density microfluidic arrays to study toxity. 6,8 By decreasing the time needed for fixation and staining, microfluidics a͒ Author to whom correspondence should be addressed.…”

Microfluidics: Emerging prospects for anti-cancer drug screening

“…These include real-time studies on a single cell level, such as timeof-flight (TOF) optophoresis and scanning thermal lens microscopy (TLM) [46][47][48][49][50] . The living cell microarrays and microfluidic cell arrays are yet other examples of emerging LOC technologies that provide important technological advances in the spatiotemporal control of biomolecules and cells [6,10,[51][52][53][54][55][56] . Pioneering microfluidic cell arrays allow collection of real-time and multiparameter data obtained from functional cell-based assays [6,10,56] .…”

“…The living cell microarrays and microfluidic cell arrays are yet other examples of emerging LOC technologies that provide important technological advances in the spatiotemporal control of biomolecules and cells [6,10,[51][52][53][54][55][56] . Pioneering microfluidic cell arrays allow collection of real-time and multiparameter data obtained from functional cell-based assays [6,10,56] . These emerging technologies create living-cell arrays that are ideal for modeling cancer microenvironments and inherently scalable for constructing a high-throughput screening platform [52][53][54][55] .…”