Bioimprinting technologies for removal of myeloblasts from peripheral blood

Medlock, Jevan

Abstract

Acute Myeloid Leukaemia (AML) is a malignancy occurring in the bone marrow and blood whereby immature and defective blast cells are overproduced. As a genetic condition, no cure is available. The condition is traditionally managed by treatment reliant on non-specific cytotoxic chemotherapy and bone marrow transplantation. Treatment is associated with causing discomfort and mortality and is ultimately ineffective; relapse is common and survival rates are poor.
Bioimprinting is a technology whereby the size, shape and morphology of biological templates are recreated in polymer matrices. Studies aim to mimic and exploit specific binding reliant on complementary size and shape interactions as seen in a number of biological processes. The field has developed from the templating of rudimentary macromolecules to whole cells with extracellular features accurate on a nanometre scale.
This study aimed to fabricate AML specific bioimprints able to discriminate neoplastic cells from patient aspirate. Myeloblasts provide an ideal target due to their inherent size difference and morphological irregularity. Bioimprints incorporated into a high throughput device could provide a vehicle for selectivity of myeloblasts, yielding an alternate treatment pathway in reducing the leukaemic burden in AML sufferers.
Herein, methods were devised and evaluated to reliably fabricate high quality bioimprints, representative of the templated cell. Key in the protocol design is the control over the proportion of the cell surface exposed to the curing polymer matrix which dictates the size of the cavities produced and in-turn the ability of uptake of target cells to the bioimprint substrates. This method should be compatible with roll-to-roll nanoimprint lithography which has been highlighted as a viable method to upscale the imprints in order to deplete very high myeloblast cell populations in AML sufferers. Bioimprints of various cell types and polymer particles of similar size were made and further used to produce positive imprints and subsequent negative replica imprints. Ultimately, a methodology was devised and bioimprints of an AML in vitro cultured cell line were fabricated and reproduced into an area of hundreds of square metres.
The success of bioimprinting technology was evaluated with high resolution microscopy and surface profiling; characterising bioimprinted cavities in comparison with the template cell type. Surface modifications were trialled in order to incur an attraction between substrates and incubated cell populations. A coating of weak cationic surface charge was introduced on the bioimprint surface, to attract the negative charges of extracellular groups. This interaction is amplified by an increased surface area contact, allowing binding of cells fitting flush into cavities. Cells unable to fit into cavities did not receive this attraction and remained unbound. With the intended use in mind, a method using materials approved for clinical use was found.
Once produced and functionalised, the retention of incubated cell populations was examined under flow conditions. In doing, a bespoke microfluidic device was designed in order to control the hydrodynamics experienced by the bioimprint allowing for a comparison of retention per surface modification parameters. Retention of target cells to bioimprints made using the same cell type was measured as a function of incubated cell suspension concentration; analysis confirmed cells were retained and localised to the bioimprinted cavities. This was compared to cells incubated on bioimprints produced from microparticles of the same size distribution. Significantly poorer retention was observed, indicating the importance of cell shape and cellular surface properties in bioimprint capture.
The preference of the bioimprints to the target cell type was assessed by exposure to binary cell mixtures of myeloblasts and PBMCs. Cell populations were characterised on account of size and shape and separately fluorescently labelled for identification and automated enumeration. Bioimprint selectivity towards the targeted cells (myeloblasts) was compared by the proportions of each cell type retained to the bioimprints. In each instance the bioimprint showed a preference for capture of the target cell type over the healthy control. It is anticipated that by reapplying or recirculating patient aspirate, myeloblasts can be completely depleted from samples due to the higher affinity. This effect was confirmed by comparison of the bioimprint path length on selectivity; using larger areas of bioimprint at fixed cell concentration to represent a recirculated population.

Citation

Medlock, J. Bioimprinting technologies for removal of myeloblasts from peripheral blood. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4223476

• Thesis Type: Thesis
• Deposit Date: Oct 6, 2021
• Publicly Available Date: Feb 24, 2023
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4223476
• Additional Information: Department of Chemistry, The University of Hull
• Award Date: Sep 1, 2018