Bioimprint aided cell recognition and depletion of human leukemic HL60 cells from peripheral blood

We report a large scale preparation of bioimprints of layers of cultured human leukemic HL60 cells which can perform cell shape and size recognition from a mixture with peripheral blood mononuclear cells (PBMCs). We demonstrate that the bioimprint-cell attraction combined with surface modification and flow rate control allows depletion of the HL60 cells from peripheral blood which can be used for development of alternative therapies of acute myeloid leukaemia (AML).AML is a clonal malignant proliferation of transformed, bone-marrow derived myeloid precursors. The disease is characterised by the rapid proliferation of the neoplastic cells (myeloblasts) resulting in failure of normal haematopoiesis with consequential bone marrow failure rapidly resulting in death if untreated.1–3 In the UK, overall survival is 16% 5 years from diagnosis. The prognosis is significantly worse in the elderly which is especially relevant as the majority of patients present over the age of 60 years.1,4–7 Therapy relies on 2–3 cycles of myeloablative chemotherapy followed by allogeneic stem cell transplants for a relatively small number of fit patients with poor prognostic features.8,9 This is accompanied by significant discomfort, and long therapy for AML is also associated with prolonged inpatient stays, considerable morbidity related to anaemia, sepsis and bleeding with an attributable mortality of 5–10%. The majority of patients relapse following induction of chemotherapy for AML and subsequent therapy is associated with a low probability of cure. Outcomes for AML patients have improved marginally over the past few decades, largely due to improvements in supportive care rather than dramatic improvements in the chemotherapeutic regimen's efficacy.10Bioimprinting is a promising area of materials chemistry aimed at mimicking and exploiting the lock-and-key interactions seen ubiquitously in nature.11–14 Cell recognition systems are relatively cheap and simple to produce with few stipulations on storage and shelf life when compared with biological interventions. The scope for possible targets is also much greater, being able to target polysaccharides, enzymes, aptamers, DNA sequences, antibodies and whole cells.12,15,16,21–24 Bioimprints of whole cells were first reported by Dickert et al.17 who imprinted yeast into a sol–gel matrix. When incubated with several strains of yeast, the substrates showed a high affinity to the template yeast strain. This effect was attributed to the large contact surface areas between the cells and the imprinted cavities. Other cell bioimprinting studies have progressed to cover a range of micro-organisms and human cells. Hayden et al.18 functionalised polyurethane with erythrocyte imprints, capable of discriminating between ABO blood groups. Though all cell targets possessed the same geometrical shape and size, imprints were able to discriminate on account of varied surface antigen expression. Subsequent studies were further able to discriminate cells with identical antibodies in different quantities to separate blood groups A1 and A2.19 Recent cell bioimprint studies largely focus on biosensor applications20,26 and are hindered by the small overall size of imprinted areas that can be produced which limits their applications for large scale extraction of targeted cells from cell mixtures. This research area is undergoing a rapid expansion towards using molecularly imprinted polymers as receptor mimics for selective cell recognition and sensing, and a recent review of size and shape targeting of cancer found no evidence so far of the use of cancer cell bioimprints in a therapeutic setting.11Here we utilised for the first time AML cell bioimprints on a large scale as a vehicle to selectively target myeloblasts due to the inherent size and morphological discrepancies compared to normal peripheral blood mononuclear cells (PBMCs) (see Fig. S1, ESI†). We explore AML cells bioimprinting to develop a new method for depletion of myeloblasts from peripheral blood cells by introducing selectivity via bespoke cell size and shape discrimination aided by myeloblast-bioimprint interactions. Our idea is based on incorporating AML cells-imprinted substrates into a flow-through type of device which offers an alternative method for removal of the leukemic burden directly from patient blood. Successful leukophoresis can potentially be used more frequently in the extraction of myeloblasts from peripheral blood which is critical in stabilizing AML patients with leukostasis associated with hyperleuocytosis. By reducing the number of circulating tumour cells, the likelihood of early relapse is also diminished.25HL60 is an immortalized human cell line derived from peripheral blood lymphocytes of a patient suffering from acute promyleocytic leukaemia. HL60 was used as a very good proxy for primary (patient derived) myeloblast cells throughout our study due to their availability and ease of culture. Here we show how the desired HL60 cell bioimprints were produced from HL60 cell layers. We also discuss the integration of the produced myeloblast imprint in a PDMS-based flow-through cell, in which its selectivity towards HL60 cells over PBMCs is investigated (Fig. 1). We fabricated bioimprints by impressing a layer of cultured HL60 cells with a curable polymer, which captures information on the cell shape, size and morphology. These were further casted with another polymer to create a “positive imprint” whose surface matches the original cell layer. Using roll-to-roll printing from the positive replica we produced a very large area of HL60 cell imprints. We engineered the surface of the bioimprint to have a weak attraction with the cells, which is strongly amplified when there is a shape and size match between the individual cells and the imprinted surface. Due to inherent size and morphology differences between myeloblasts and normal blood cells, this resulted in much higher retention of the former on the bioimprint. This allows their selective trapping from peripheral blood based on cell shape and size recognition, much cheaper than using surface functionalisation with a combination of specific antibodies for myeloblasts. We tested the bioimprints selectivity in a device for depleting cultured HL60 cells from healthy white blood cells. This cell recognition technology can potentially deplete myeloblasts from the blood of AML patients and provide an alternative route for inducing minimal residual disease, which is associated with reduced relapses and improved patient outcomes

Removal of Human Leukemic Cells from Peripheral Blood Mononuclear Cells by Cell Recognition Chromatography with Size Matched Particle Imprints

We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on sizematched functionalized particle imprints. Negative imprints were prepared from layers of 15 μm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia

Targeted removal of blood cancer cells from mixed cell populations by cell recognition with matching particle imprints

We report a new approach for separation of blood cancer cells from healthy white blood cells based on cell recognition by surface functionalised particle imprints. We prepared polymeric particle imprints from a layer of suspension of monodisperse PMMA microbeads which closely match the size of in vitro cultured human leukaemia cells (HL60). The imprints were replicated on a large scale with UV curable polyurethane resin using nanoimprinting lithography and surface functionalized with a cationic polymer, a branched polyethylene imine (bPEI), and a Pluronic surfactant, Poloxamer 407, to engineer a weak attraction towards the cells. The latter is amplified several orders of magnitude when a cell of a closely matching size and shape fits into the imprint cavity which multiplies the contact area between the cell surface and the imprint. The particle imprints were optimised for their specificity toward blood cancer cells by treatment with oxygen plasma and then subsequent coatings with bPEI and Poloxamer 407 with various functionalisation concentrations. We tested the surface functionalised imprints for their specificity in retaining in vitro cultured human leukaemic cells (HL60) over healthy human peripheral blood mononuclear cells (PBMCs) in a flow through chamber. The effect of the flushing flow rate of the mixed cell suspension over the particle imprint and the imprint length were also investigated. At each step the selectivity towards HL60 was assessed. Selective isolation of an increased amount of HL60 tumour cells over PBMC was ultimately achieved as a function of the cell seeding ratio on the particle imprint. The effect is attributed to the substantial size difference between the HL60 cell and the PBMCs. The data presented show that relatively inexpensive PMMA microbeads imprints can be utilised as a cell separation technique which could ultimately lead to novel therapies for removal of neoplastic cells from the peripheral blood of acute myeloid leukaemia patients

Roll-to-Roll pilot line for large-scale manufacturing of microfluidic devices

Roll-to-roll (R2R) technologies with roller-based nanoimprinting methods enable manufacturing of highly cost-effective and large-scale sheets of flexible polymer film with precise structures on a micro- and nanoscale 1. Areas that can benefit strongly from such large scale technologies are microfluidics, biosensors, and lab-on-chip products for point of care diagnostics, drug discovery and food control. Here, R2R fabrication could greatly reduce production costs and increase manufacturing capacity with respect to currently used products. A pilot line with this technology is investigated in the European Horizon 2020 project R2R Biofluidics and its capabilities are tested on two Demonstrators: - Demonstrator 1: In-vitro diagnostic chip with imprinted microfluidic channels based on optical chemiluminescence measurement by photodetectors. - Demonstrator 2: Neuronal cell culture plate with imprinted cavities and channels for controlled culturing and fluorescence imaging of neurons, for high throughput drug screening. Please click Additional Files below to see the full abstract

High-throughput roll-to-roll production of polymer biochips for multiplexed DNA detection in point-of-care diagnostics

Roll-to-roll UV nanoimprint lithography has superior advantages for high-throughput manufacturing of micro- or nano-structures on flexible polymer foils with various geometries and configurations. Our pilot line provides large-scale structure imprinting for cost-effective polymer biochips (4500 biochips/hour), enabling rapid and multiplexed detections. A complete high-volume process chain of the technology for producing structures like μ-sized, triangular optical out-couplers or capillary channels (width: from 1 μm to 2 mm, height: from 200 nm up to 100 μm) to obtain biochips (width: 25 mm, length: 75 mm, height: 100 μm to 1.5 mm) was described. The imprinting process was performed with custom-developed resins on polymer foils with resin thicknesses ranging between 125–190 μm. The produced chips were tested in a commercial point-of-care diagnostic system for multiplexed DNA analysis of methicillin resistant Staphylococcus aureus (e.g., mecA, mecC gene detections). Specific target DNA capturing was based on hybridisation between surface bound DNA probes and biotinylated targets from the sample. The immobilised biotinylated targets subsequently bind streptavidin–horseradish peroxidase conjugates, which in turn generate light upon incubation with a chemiluminescent substrate. To enhance the light out-coupling thus to improve the system performance, optical structures were integrated into the design. The limits-of-detection of mecA (25 bp) for chips with and without structures were calculated as 0.06 and 0.07 μM, respectively. Further, foil-based chips with fluidic channels were DNA functionalised in our roll-to-roll micro-array spotter following the imprinting. This straightforward approach of sequential imprinting and multiplexed DNA functionalisation on a single foil was also realised for the first time. The corresponding foil-based chips were able to detect mecA gene DNA sequences down to a 0.25 μM concentration.This research was supported by R2R BIOFLUIDICS project (http://www.r2r-biofluidics.eu/) under Horizon 2020 European Union (EU) Research and Innovation Programme with grant agreement no 646260. The research was also partially supported by NextGenMicrofluidics project (https:// www. nextgenmicrofluidics.eu/) under HORIZON2020 with grant agreement no 862092. The authors cordially thank Gerburg Schider & Gerhard Mohr, Markus Postl, Paul Patter and Alexander Wheeldon (JOANNEUM RESEARCH – Materials, Weiz, Austria) for revising the manuscript, preparing all the chip and R2R pilot line illustrations, taking the photographs and providing technical support, respectively. The authors are also grateful to Christian Wolf and Johannes Götz (JOANNEUM RESEARCH – Materials, Weiz, Austria) for their supports in the fluidic design and R2R UV-NIL structuring, respectively. We further kindly thank Alba Simon Munoz and Robert Fay (SCIENION AG, Berlin, Germany) for providing the illustration of the R2R micro-spotting line. PT specially thanks Ege Ozgun (NANOTAM, Bilkent University, Ankara, Turkey) for critically reading the manuscript

Tactile sensitivity is reduced in mild cognitive impairment

Sensory impairment has been related to age-associated cognitive decline. While these associations were investigated primarily in the auditory and visual domain, other senses such as touch have rarely been studied. Thus, it remains open whether these results are specific for particular sensory domains, or rather point to a fundamental role of sensory deficits in cognitive decline. In this study, data from 31 participants with mild cognitive impairment (MCI), 46 participants with frailty, and 23 non-clinical control participants (NCCs) were included. We assessed sensory function using visual acuity, absolute hearing threshold, and mechanical detection threshold. Cognitive function in participants with MCI was assessed using associative memory performance. Group differences on sensory thresholds were tested using analyses of covariance with age, gender, and years of education entered as covariates. Associations between measures within the MCI group were evaluated using Spearman correlations. We found a significant difference in tactile detection threshold between the groups (F2,93 = 10·35, p < 0·001, η2 = 0·18). Participants with MCI showed significantly reduced tactile acuity compared to participants with frailty (p < 0·001) and NCCs (p = 0·024). In the MCI sample, lower associative memory performance was significantly related to reduced tactile (rs = 0·39, p = 0·031) and auditory acuity (rs = 0·41, p = 0·022). Our results indicate that sensory deficits other than in the auditory and visual domain might contribute to cognitive decline. Prospective studies should investigate the age-related alterations of multimodal sensory processes and its contribution to dementia-related processes

Enabling large area and high throughput roll-to-roll NIL by novel inkjetable and photo-curable NIL-resists

The high throughput and large area nanostructuring of flexible substrates by continuous roller processes has great potential for future custom applications like wire grid polarizers, antireflection films, or super-hydrophobic surfaces. For each application different material characteristics have to be considered, e.g. refractive index, hydrophobicity, or dry etch stability. Herein, we show experimental results of nanoimprint lithography resist developments focused on inkjetable and photo-curable resists suitable for high throughput production, especially roll-to-roll NIL. The inkjet deposition of the novel materials is demonstrated by the use of different state-of-the-art inkjet printheads at room temperature. A plate-to-plate process on silicon substrates was successfully implemented on a NPS300 nano patterning stepper with previously inkjet dispensed NIL resist. Furthermore, we demonstrate a throughput of 30 m/min in a roller NIL process on PET. Dry etching of unstructured thin films on Si wafers was performed, and it was demonstrated that the etch stability in Si is tunable to a value of 3.5:1 by a concise selection of the resist components. The surface roughness of the etched films was measured to be < 2 nm, after etching of around 100 nm of the resist films what is an essential factor for a low line edge roughness. All resists reported herein can be deposited via inkjet dispensing at room temperature, are suitable for continuous high throughput imprinting on flexible substrates, and are applicable in step-wise NIL processes with good etch resistance in dry etch processes

High Throughput Roll-to-Roll Production of Microfluidic Chips

A high throughput manufacturing process of microfluidic chips based on Roll-to-Roll imprinting is presented. With this procedure, microfluidic patterns can be produced on large area polymer substrates. The subsequent steps of inlet drilling, bonding and electrode printing are set-up on large area processes, too. Overall, this strategy allows highly parallelized processing of large numbers of chips—all costly steps of individual chip handling are avoided. The chips were used for the characterization of inorganic ions for soil nutrient analysis