To improve the survival rate of cancer patients, new diagnosis strategies are necessary to detect lower levels of cancer cells before and after treatment regimens. The scarcity of diseased cells, particularly in residual disease after treatment, demands highly sensitive detection approaches or the ability to enrich the diseased cells in relation to normal cells. We report a label-free microfluidic approach to enrich leukemia cells from healthy cells using inherent differences in cell biophysical properties. The microfluidic device consists of a channel with an array of diagonal ridges that recurrently compress and translate flowing cells in proportion to cell stiffness. Using devices optimized for acute T cell leukemia model Jurkat, the stiffer white blood cells were translated orthogonally to the channel length, while softer leukemia cells followed hydrodynamic flow. The device enriched Jurkat leukemia cells from white blood cells with an enrichment factor of over 760. The sensitivity, specificity, and accuracy of the device were found to be >0.8. The values of sensitivity and specificity could be adjusted by selecting one or multiple outlets for analysis. We demonstrate that low levels of Jurkat leukemia cells (1 in 104 white blood cells) could be more quickly detected using flow cytometry by using the stiffness sorting pre-enrichment. In a second mode of operation, the device was implemented to sort resistive leukemia cells from both drug-sensitive leukemia cells and normal white blood cells. Therefore, microfluidic biomechanical sorting can be a useful tool to enrich leukemia cells that may improve downstream analyses.

Efficient intracellular delivery of target macromolecules remains a major obstacle in cell engineering and other biomedical applications. We discovered a unique cell biophysical phenomenon of transient cell volume exchange by using microfluidics to rapidly and repeatedly compress cells. This behavior consists of brief, mechanically induced cell volume loss followed by rapid volume recovery. We harness this behavior for high-throughput, convective intracellular delivery of large polysaccharides (2000 kDa), particles (100 nm), and plasmids while maintaining high cell viability. Successful proof of concept experiments in transfection and intracellular labeling demonstrated potential to overcome the most prohibitive challenges in intracellular delivery for cell engineering.

Cancers consist of a heterogeneous populations of cells that may respond differently to treatment through drug-resistant sub-populations. The scarcity of these resistant sub-populations makes it challenging to understand how to counter their resistance. We report a label-free microfluidic approach to separate cancer cells treated with chemotherapy into sub-populations enriched in chemoresistant and chemosensitive cells based on the differences in cellular stiffness. The sorting approach enabled analysis of the molecular distinctions between resistant and sensitive cells. Consequently, the role of multiple mechanisms of drug resistance was identified, including decreased sensitivity to apoptosis, enhanced metabolism, and extrusion of drugs, and, for the first time, the role of estrogen receptor in drug resistance of leukemia cells. To validate these findings, several inhibitors for the identified resistance pathways were tested with chemotherapy to increase cytotoxicity sevenfold. Thus, microfluidic sorting can identify molecular mechanisms of drug resistance to examine heterogeneous responses of cancers to therapies.

The enrichment of viable cells is an essential step to obtain effective products for cell therapy. While procedures exist to characterize the viability of cells, most methods to exclude nonviable cells require the use of density gradient centrifugation or antibody-based cell sorting with molecular labels of cell viability. We report a label-free microfluidic technique to separate live and dead cells that exploits differences in cellular stiffness. The device uses a channel with repeated ridges that are diagonal with respect to the direction of cell flow. Stiff nonviable cells directed through the channel are compressed and translated orthogonally to the channel length, while soft live cells follow hydrodynamic flow. As a proof of concept, Jurkat cells are enriched to high purity of viable cells by a factor of 185-fold. Cell stiffness was validated as a sorting parameter as nonviable cells were substantially stiffer than live cells. To highlight the utility for hematopoietic stem cell transplantation, frozen samples of cord blood were thawed and the purity of viable nucleated cells was increased from 65% to over 94% with a recovery of 73% of the viable cells. Thus, the microfluidic stiffness sorting can simply and efficiently obtain highly pure populations of viable cells.

Cell surface molecular adhesions govern many important physiological processes and are used to identify cells for analysis and purifications. But most effective cell adhesion separation technologies use labels or long-term attachments in their application. While label-free separation microsystems typically separate cells by size, stiffness, and shape, they often do not provide sufficient specificity to cell type that can be obtained from molecular expression. We demonstrate a label-free microfluidic approach capable of high throughput separation of cells based upon surface molecule adhesion. Cells are flowed through a microchannel designed with angled ridges at the top of the channel and coated with adhesive ligands specific to target cell receptors. The ridges slightly compress passing cells such that adhesive contact can be made with sufficient surface area without unduly affecting cell trajectories because of cell stiffness. Thus, sorting is sensitive to cell adhesion but not to stiffness or cell size. The enforced interactions between the cells and the ridges ensure that a high flow rate can be used without lift forces quenching adhesion. As a proof of principle of the method, we separate both Jurkat and HL60 cell lines based on their differential expression of PSGL-1 ligand by using a ridged channel coated with P selectin. We demonstrate 26-fold and 3.8-fold enrichment of PSGL-1 positive and 4.4-fold and 3.2-fold enrichment of PSGL-1 negative Jurkat and HL60 cells, respectively. Increasing the number of outlets to five allows for greater resolution in PSGL-1 selection resulting in fractionation of a single cell type into subpopulations of cells with high, moderate, and low PSGL-1 expression. The cells can flow at a rate of up to 0.2 m/s, which corresponds to 0.045 million cells per minute at the designed geometry, which is over 2 orders of magnitude higher than previous adhesive-based sorting approaches. Because of the short interaction time of the cells with the adhesive surfaces, the sorting method does not further activate the cells due to molecular binding. Such an approach may find use in label-free selection of cells for a highly expressed molecular phenotype.

Traumatic injury to the central nervous system is a significant health problem. There is no effective treatment available partly because of the complexity of the system. Implementation of multifunctional micro-and nano-device based combinatorial therapeutics can provide biocompatible and tunable approaches to perform on-demand release of specific drugs. This can help the damaged cells to improve neuronal survival, regeneration of axons, and their reconnection to appropriate targets. Nano-topological features induced rapid cell growth is especially important towards the design of effective platforms to facilitate damaged neural circuit reconstruction. In this study, for the first time, feasibility of neuron-like PC12 cell growth on untreated and easy to prepare nanotextured surfaces has been carried out. The PC12 neuron-like cells were cultured on micro reactive ion etched. nanotextured glass coverslips. The effect of nanotextured topology as physical cue for the growth of PC12 cells was observed exclusively, eliminating the possible influence(s) of the enhanced concentration of coated materials on the surface. The cell density was observed to increase by almost 200% on nanotextured coverslips compared to plain coverslips. The morphology study indicated that PC12 cell attachment and growth on the nanotextured substrates did not launch any apoptotic machinery of the cell. Less than 5% cells deformed and depicted condensed nuclei with apoptotic bodies on nanotextured surfaces which is typical for the normal cell handling and culture. Enhanced PC12 cell proliferation by such novel and easy to prepare substrates is not only attractive for neurite outgrowth and guidance, but may be used to increase the affinity of similar cancerous cells (ex: B35 neuroblastoma) and rapid proliferation thereafter-towards the development of combinatorial theranostics to diagnose and treat aggressive cancers like neuroblastoma.

Detection of circulating tumor cells (CTCs) in the early stages of cancer is a great challenge because of their exceedingly small concentration. There are only a few approaches sensitive enough to differentiate tumor cells from the plethora of other cells in a sample like blood. In order to detect CTCs, several antibodies and aptamers have already shown high affinity. Nanotexture can be used to mimic basement membrane to further enhance this affinity. This article reports an approach to fabricate nanotextured polydimethylsiloxane (PDMS) substrates using micro reactive ion etching (micro-RIE). Three recipes were used to prepare nanotextured PDMS using oxygen and carbon tetrafluoride. Micro-RIE provided better control on surface properties. Nanotexturing improved the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers against cell membrane overexpressed with epidermal growth factor receptors. In all cases, nanotexture of PDMS increased the effective surface area by creating nanoscale roughness on the surface. Nanotexture also enhanced the growth rate of cultured cells compared to plain surfaces. A comparison among the three nanotextured surfaces demonstrated an almost linear relationship between the surface roughness and density of captured tumor cells. The nanotextured PDMS mimicked biophysical environments for cells to grow faster. This can have many implications in microfluidic platforms used for cell handling.

Microfluidic channels have been implemented to detect cancer cells from blood using electrical measurement of each single cell from the sample. Every cell provided characteristic current profile based on its mechano-physical properties. Cancer cells not only showed higher translocation time and peak amplitude compared to blood cells, their pulse shape was also distinctively different. Prevalent microfluidic channels are plain but we created nanotexture on the channel walls using micro reactive ion etching (micro-RIE). The translocation behaviors of the metastatic renal cancer cells through plain and nanotextured PDMS microchannels showed clear differences. Nanotexture enhanced the cell-surface interactions and more than 50% tumor cells exhibited slower translocation through nanotextured channels compared to plain devices. On the other hand, most of the blood cells had very similar characteristics in both channels. Only 7.63% blood cells had slower translocation in nanotextured microchannels. The tumor cell detection efficiency from whole blood increased by 14% in nanotextured microchannels compared to plain channels. This interesting effect of nanotexture on translocation behavior of tumor cells is important for the early detection of cancer.

Aims: Circulating tumor cells (CTCs) have significant diagnostic value for cancer patients. We report a label -free, simple and rapid microchannel filter type device for isolation ofknown metastatic cancercells based on their mechano-physical properties like size and deformability. Study Design:Metastatic renal cancer cells were highly elastic and squeeze d through microchannels much smaller than their size. Using a reverse -selectivity approach, the number of microchannels and their dimensionswere varied to optimize and reduce the shear stress o n tumor cells such that these did

Cell adhesion, morphology and growth are known to be influenced by microscale topography of the surfaces. Taking this a step further, nanotextured polydimethylsiloxane (PDMS) substrates were fabricated by micro reactive ion etching (Micro-RIE). This simple process gave good control on surface texture to improve the affinity of PDMS to enhance cell growth. The effects of various nanoscale roughnesses on cell growth were also quantified. Three different recipes were used to prepare nanotextured PDMS by micro-RIE using oxygen (O2) and carbon tetrafluoride (CF4). First of all etching was performed for 12 min and flow rate of O2 and CF4 was kept at a ratio of 1:1 to fabricate surface 1. Surface 2 was fabricated just by changing the ratio of O2 and CF4 to 1:2. Then the flow rates of gases were kept same as recipe 1 but the etching time was increased to 25 min to prepare surface 3. Next, surface topography of these PDMS substrates was evaluated quantitatively by measuring root mean square surface roughness using an atomic force microscope (AFM). The measured average roughness of three nanotextured PDMS surfaces were found 242.92±87.709 nm, 515±102.28 nm and 629.17±109.67 nm for surface 1, surface 2 and surface 3 respectively. The roughnesses of three types of nanotextured surfaces showed a correlation on cell growth. The cell growth rate on flat PDMS surfaces is known to be very low but nanotextured PDMS surfaces allowed exciting cell growth for three different types of cells (human glioblastoma (hGBM), astrocytesand fibroblast cells).The cells were seeded on various surfaces coated with laminin and incubated for 3 days at 37oC. The cell densities of experimental surfaces were calculated from their images using ImageJ software. For all types of cells, growth was higher on nantoextured PDMS surfaces compare to flat PDMS surface and surface 3 exhibited highest cell density among three nanotextured surfaces. However density of astrocytes cells was 114 ± 16.15 cells per mm2 in surface 3 where as flat PDMS had only 28 ± 3.58 cells per mm2. But standard well plate had higher cell density compare to surface 3 and it was 140 ± 18.07 cells per mm2. Again density of fibroblasts cells in surface 3 was 272.80 ± 13.68 cells per mm2 which was higher compare to flat PDMS but lower than standard well plate. On the other hand surface 3 exhibited very similar cell density as standard well plate for hGBM cells and cell densities were 346 ± 70.64 cells per mm2 and 334.67 ± 63.23 cells per mm2 respectively. Consequently it can be deduced that cell growth strongly depends on surface nanotexturing. By comparing three nanotextured surfaces it was found that surface 1 exhibited lowest roughness compare to surface 2 and surface 3. Therefore available surface area for laminin attachment was lowest in this surface. But surface 2 and surface 3 had higher roughness and therefore provided larger surface area to attach more laminin. As cell growth is a direct function to available laminin attached in the surface therefore cell density was more in later surfaces. In addition to that circulating tumor cells (CTCs) are known to have very high affinity towards nanotextured surfaces. Therefore density of only hGBM cells in nanotextured surface was very similar to cell density in standard well plate. As a result nanotextured PDMS may be thus useful for cancer cytology devices, especially to culture isolated CTCs for further investigation in microfluidic platforms.

This paper presents a simple approach to create a two-tiered surface for superior cancer cell isolation. The idea is inspired by the interactions of cells with a nanotextured basement membrane. The texture mimicked the extracellular matrix and basement membrane for superior target cell adhesion. Prepared micro+nanotextured surfaces showed enhanced cell capture. Preparation of the two-tiered surface was done using micro- and nanotexturing and was easily reproducible. It has been shown before that the larger surface area of a nanotextured surface assists the cell's attachment through surface-anchored ligands. Taking it a step further, ligand functionalized two-level micro+nanotextured surfaces improved the sensitivity of the cancer cell isolation over simple flat nanotexturing. The isolation efficiency increased by 208% compared to the surface with a single-level nanotexture. The two-tiered surface was compatible with previously reported nanotextured devices used for cancer cell isolation. Micro-texture on the glass surface was created using simple sand gritting, followed by reactive ion etching (RIE) of the entire surface. The approach could create large surface areas within a short time while maintaining superior cell isolation efficiency.

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are widely used for controlled delivery of bioactive agents in therapeutic applications. These nanoparticles show bioavailability, better encapsulation, controlled release, biocompatibility, and in vivo biodegradability. This paper reports a novel approach to synthesize porous PLGA nanoparticles and their use as controlled release vehicles. Bovine serum albumin (BSA) loaded PLGA nanoparticles (porous and nonporous) were synthesized using water-in-oil-in-water double emulsion method. Specifically, PLGA nanoparticles were prepared using chloroform and polyvinyl alcohol, and freeze drying was employed for the phase separation to obtain the nanoparticles. The porous nanoparticles were prepared through the salt-leaching process where sodium bicarbonate was used as an extractable porogen. In vitro drug release behavior of porous and nonporous nanoparticles was monitored over a period of 30 days. A much more enhanced BSA release was observed in case of porous polymeric nanoparticles when compared to nonporous nanoparticles. The characterization was done using laser scattering, zeta potential analysis, and scanning electron microscopy. The drug loading efficiencies for BSA in porous and nonporous PLGA nanoparticles were 65.50% and 77.59%, respectively. Over a period of 30 days, the cumulative BSA released from PLGA porous and nonporous nanoparticles were measured to be 87.41% and 59.91%, respectively. The synthesis of porous nanoparticles with this novel, rapid, and inexpensive method opens a new horizon of using a wide range of cheap and easily-accessible water-soluble salts that can be extracted through leaching process to introduce porous morphology on the nanoparticle surfaces. The porous nanoparticles can have useful applications in controlled drug delivery systems.

Curcumin and bovine serum albumin (BSA) were used as model drugs and loaded into microand nanoparticles of biodegradable poly(lactic-co-glycolic acid) (PLGA). The PLGA was incorporated into hydrophilic and biocompatible gelatin scaffolds to design a controlled drug release system. The gelatin scaffolds were cross-linked using glutaraldehyde. The controlled delivery of drugs from biologically active PLGA microand nanoparticles was measured and these showed consistent release for 30 days. Curcuminand BSA-loaded PLGA micro/nanoparticles-based gelatin scaffolds define a novel approach to embed multiple drug molecules to overcome multidrug resistance as well as depict a new type of biocompatible and biodegradable implant. Such scaffold constructs can be used for breast implants after lumpectomy to not only overcome cosmetic issues, but also to provide sustained drug release during healing process. In one type of construct, only BSA-loaded microparticles were mixed with gelatin, while in the other type of construct, both BSAand curcumin-loaded PLGA microparticles were embedded. BSAand curcumin-loaded nanoparticles were also embedded into gelatin constructs to see the effects of particle size on drug release. After 30 days, cumulative BSA release from PLGA microand nanoparticles embedded in gelatin scaffold were measured to be 69.87% and 86.11%, respectively. The cumulative release of curcumin was measured to be 53.11% and 60.42% from curcumin-loaded PLGA microand nanoparticles, respectively. A statistically significant difference was seen in cumulative drug release from these scaffolds (p value <; 0.05).