Papers by Kuldeepsinh Rana
Small, Jul 18, 2017
Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, ... more Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (μwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 μg mL−1 tPA. μWheels following corkscrew trajectories can also penetrate through 100 μm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal.
Biotechnology and Bioengineering, Apr 15, 2009
The survival rate for patients with metastases versus localized cancer is dramatically reduced, w... more The survival rate for patients with metastases versus localized cancer is dramatically reduced, with most deaths being associated with the formation of secondary tumors. Circulating cancer cells interact with the endothelial lining of the vasculature via a series of adhesive interactions that facilitate tethering and firm adhesion of cancer cells in the initial steps of metastasis. TNF‐related apoptosis‐inducing ligand (TRAIL) holds promise as a tumor‐specific cancer therapeutic, by inducing a death signal by apoptosis via the caspase pathway. In this study, we exploit this phenomenon to deliver a receptor‐mediated apoptosis signal to leukemic cells adhesively rolling along a TRAIL and selectin‐bearing surface. Results show that cancer cells exhibit selectin‐mediated rolling in capillary flow chambers, and that the rolling velocities can be controlled by varying the selectin and selectin surface density and the applied shear stress. It was determined that a 1 h rolling exposure to a functionalized TRAIL and E‐selectin surface was sufficient to kill 30% of captured cells compared to static conditions in which 4 h exposure was necessary to kill 30% of the cells. Thus, we conclude that rolling delivery is more effective than static exposure to a TRAIL immobilized surface. We have also verified that there is no significant effect of TRAIL on hematopoietic stem cells and other normal blood cells. This represents the first demonstration of a novel biomimetic method to capture metastatic cells from circulation and deliver an apoptotic signal. Biotechnol. Bioeng. 2009;102: 1692–1702. © 2008 Wiley Periodicals, Inc.
Biomicrofluidics, Sep 1, 2014
Despite the myriad of soft lithography based micropatterning methods available to researchers, it... more Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10-100 lm) that are spaced far apart (1-10 mm). In this report, we describe a combined microfluidicmicrostencil patterning method that can produce multifunctional substrates of small features, O(10 lm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10-100 lm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay. V
Biomicrofluidics, Nov 1, 2015
In vitro assays of platelet function and coagulation are typically performed in the presence of a... more In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.
Blood Reviews, Sep 1, 2016
Blood flow regulates coagulation and fibrin formation by controlling the transport, or mass trans... more Blood flow regulates coagulation and fibrin formation by controlling the transport, or mass transfer, of zymogens, co-factors, enzymes, and inhibitors to, from, and within a growing thrombus. The rate of mass transfer of these solutes relative to their consumption or production by coagulation reactions determines, in part, the rate of thrombin generation, fibrin deposition, and thrombi growth. Experimental studies on the influence of blood flow on specific coagulation reactions are reviewed here, along with a theoretical framework that predicts how flow influences surface-bound coagulation binding and enzymatic reactions. These flow-mediated transport mechanisms are also used to interpret the role of binding site densities and injury size on initiating coagulation and fibrin deposition. The importance of transport of coagulation proteins within the interstitial spaces of thrombi is shown to influence thrombi architecture, growth, and arrest.
Cellular and Molecular Bioengineering, Oct 24, 2016
Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The ty... more Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The type of thrombus that forms depends on the nature of the injury and hemodynamics. There are many models of intravascular thrombus formation whereby blood is exposed to prothrombotic molecules on a solid substrate. However, there are few models of extravascular thrombus formation whereby blood escapes into the extravascular space through a hole in the vessel wall. Here, we describe a microfluidic model of hemostasis that includes vascular, vessel wall, and extravascular compartments. Type I collagen and tissue factor, which support platelet adhesion and initiate coagulation, respectively, were adsorbed to the wall of the injury channel and act synergistically to yield a stable thrombus that stops blood loss into the extravascular compartment in~7.5 min. Inhibiting factor VIII to mimic hemophilia A results in an unstable thrombus that was unable to close the injury. Treatment with a P2Y12 antagonist to reduce platelet activation prolonged the closure time twofold compared to controls. Taken together, these data demonstrate a hemostatic model that is sensitive to both coagulation and platelet function and can be used to study coagulopathies and platelet dysfunction that result in excessive blood loss.
Journal of Bionic Engineering, Dec 1, 2009
Circulating Tumor Cells (CTC) have the potential to be used clinically as a diagnostic tool and a... more Circulating Tumor Cells (CTC) have the potential to be used clinically as a diagnostic tool and a treatment tool in the field of oncology. As a diagnostic tool, CTC may be used to indicate the presence of a tumor before it is large enough to cause noticeable symptoms. As a treatment tool, CTC isolated from patients may be used to test the efficacy of chemotherapy options to personalize patient treatment. One way for tumors to spread is through metastasis via the circulatory system. CTC are able to exploit the natural leukocyte recruitment process that is initially mediated by rolling on transient selectin bonds. Our capture devices take advantage of this naturally occurring recruitment step to isolate CTC from whole blood by flowing samples through selectin and antibody-coated microtubes. Whole blood was spiked with a known concentration of labeled cancer cells and then perfused through pre-coated microtubes. Microtubes were then rinsed to remove unbound cells and the number of labeled cells captured on the lumen was assessed. CTC were successfully captured from whole blood at a clinically relevant level on the order of 10 cells per mL. Combination tubes with selectin and antibody coated surface exhibited higher capture rate than tubes coated with selectin alone or antibody alone. Additionally, CTC capture was demonstrated with the KG1a hematopoietic cell line and the DU145 epithelial cell line. Thus, the in vivo process of selectin-mediated CTC recruitment to distant vessel walls can be used in vitro to target CTC to a tube lumen. The biomolecular coatings can also be used to capture CTC of hematopoietic and epithelial tumor origin and is demonstrated to sensitivities down to the order of 10 CTC per mL. In a related study aimed at reducing the blood borne metastatic cancer load, we have shown that cells captured to a surface can be neutralized by a receptor-mediated biochemical signal. In the proposed method we have shown that using a combined selectin and TRAIL (TNF Related Apoptosis Inducing Ligand or Apo 2L) functionalized surface we are able to kill about 30% of the captured cells in a short duration of one hour whereas it took about 4 hours to kill the same proportion of cells without flow on a similarly functionalized surface. Here we have taken the approach a step further by showing that with very small doses of chemotherapeutic agents like bortezomib, we can increase the kill rate of CTC, thus allowing the device to function in scenarios where the patient is undergoing treatment. We show here that, with leukemic cells treated with bortezomib we are able to kill about 41% of the captured cells.
Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2014
Blood, Dec 7, 2017
Fibrinolytic drugs such as recombinant tissue plasminogen activator (tPA) have been successfully ... more Fibrinolytic drugs such as recombinant tissue plasminogen activator (tPA) have been successfully used to reestablish blood flow in thrombotic strokes. However, they can also lead to intracranial hemorrhaging, must be used within hours of the onset of symptoms, and have limited efficacy in small vessel occlusions distal to major cerebral arteries. Alternatively, catheter-based thrombectomy devices can restore blood flow quickly but are invasive, limited to larger arteries, and can leave residual prothrombotic material on vessel walls. An approach that overcomes these limitations is the injection of individual superparamagnetic particles coated with fibrinolytic drugs that, upon application of a rotating magnetic field, self-assemble into microbots we call microwheels capable of translating to and directly attacking thrombi. We describe an approach that uses the vessel wall to propel in situ assembled microwheels via rolling.1 Driven by low-strength external magnetic fields (~10 mT), microwheel movement occurs with greatly improved speeds (>100 µm/sec) and directional control compared to approaches that use magnetic field gradients to direct similar types of microdevices. Using this approach, tPA-functionalized particles are introduced at a subtherapeutic concentration, which accumulate to therapeutic levels at the periphery of a thrombus. Fibrinolytic microwheels bore into fibrin-rich and platelet-rich thrombi by mechanical action and accomplish reperfusion faster than soluble tPA at comparable concentrations.2 For example, fibrinolysis of plasma-derived fibrin gels is five-fold faster using tPA-microwheels compared to 1 µg/mL tPA. Using a microfluidic model of vascular injury, ~100 µm sized platelet-rich thrombi were formed on collagen-tissue factor surfaces and then ablated with tPA-microwheels in less than five minutes. Combining mechanical and biochemical methods, this approach could reduce the risk of bleeding associated with fibrinolytics and broaden their indications. 1. Tasci TO, Herson PS, Neeves KB, Marr DWM. Surface-enabled propulsion and control of colloidal microwheels. Nature Communication, 2016;7:10225. 2. Tasci TO, Disharoon D, Schoeman RM, Rana K, Herson PS, Marr DWM, Neeves KB. Enhanced fibrinolysis with magnetically powered colloidal microwheels. Small;in press Disclosures Neeves: Colorado School of Mines: Patents & Royalties.
Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mountin... more Metastasis through the bloodstream contributes to poor prognosis in many types of cancer. Mounting evidence implicates selectin-based adhesive interactions between cancer cells and the blood vessel wall as facilitating this process, in a manner similar to leukocyte trafficking during inflammation. Here, we describe a unique approach to target and kill colon and prostate cancer cells in the blood that causes circulating leukocytes to present the cancer-specific apoptosis ligand TRAIL on their surface along with E-selectin adhesion receptor. This approach, demonstrated in vitro with human blood and also in mice, mimics the cytotoxic activity of natural killer cells and increases the surface area available for delivery of the receptor-mediated signal. The resulting “unnatural killer cells” hold promise as an effective means to neutralize circulating tumor cells that enter blood with the potential to form new metastases.
British Journal of Haematology, Mar 1, 2008
Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of... more Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34-positive (CD34 +) HSPCs using monolayers of the cell adhesion protein P-selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm 2. After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34 + cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed-loop arterio-venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst-forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.
In May 2002, he received his Bachelor's of Engineering from the Department of Chemical Engineerin... more In May 2002, he received his Bachelor's of Engineering from the Department of Chemical Engineering with distinction. Following the bachelor's degree, the author worked with a pharmaceuticals intermediate manufacturer in Vadodara as a process development engineer.
In previous work, we have described the adhesive capture of circulating stem cells to surfaces co... more In previous work, we have described the adhesive capture of circulating stem cells to surfaces coated with adhesive selectin protein, both in vitro and in vivo. Here we describe PDMS surfaces microfabricated to contain an array of square 80 × 80 × 80 micron cavities. These cavities are intended to provide a local bioreactor environment to culture stem cells over extended periods of time, while sheltered from the higher shear stresses of the surrounding blood flow external of the cavities. In this paper we present in vitro flow experiments with polymeric, blood cell-sized microspheres, showing the creation of stable vortices within the microscale cavities. Computational fluid dynamics (CFD) was performed to predict the velocity field within the cavity, and for comparison with experimentally determined microsphere velocities. Future work will establish the ability to place local chemoattract molecules within the cavity interior, and the ability to accumulate viable stem cells within these cavities.
Investigative Ophthalmology & Visual Science, 2010
Scientific Reports, Apr 26, 2013
A physical sciences network characterization of non-tumorigenic and metastatic cells The Physical... more A physical sciences network characterization of non-tumorigenic and metastatic cells The Physical Sciences-Oncology Centers Network* To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis. T he conversion from a non-tumorigenic state to a metastatic one is of critical interest in cancer cell biology, as most deaths from cancer occur due to metastasis 1. Typically, we think of the activation of metastasis as one of the hallmarks of cancer 2 and as a highly regulated, multistep process defined by a loss of cell adhesion due to reduced expression of cell adhesion molecules such as E-cadherin, degradation of the extracellular matrix (ECM), conversion to a motile phenotype, vascular infiltration, exit and colonization to a new organ site (i.e., intra-and extravasation), dormancy, and re-activation. From a physical sciences perspective, metastasis can be viewed as a ''phase'' transition, albeit occuring far from thermodynamic equilibrium 3. Though this transition has been the focus of much cancer biology research, there is still an incomplete understanding of this phase change, in particular, the physical biology of the metastatic state of a cell compared to its pre-malignant state. Understanding the physical forces that metastatic cells experience and overcome in their microenvironment may improve our ability to target this key step in tumor progression. The newly formed Physical Sciences-Oncology Centers (PS-OC) Network, sponsored by and under the auspices of the Office of Physical Sciences-Oncology at the National Cancer Institute (OPSO/NCI), is a multidisciplinary network of twelve research centers across the US formed, in part, to test the fundamental hypothesis that physical processes (e.g., mechanics, dynamics) play a critical role in cancer initiation and metastasis. The PS-OC Network brings analytic techniques and perspectives from the physical sciences to the interpretation of biological data and consists of physicists, engineers, mathematicians, chemists, cancer biologists, and computational scientists. The goal of the PS-OC Network is to better understand the physical and chemical forces that shape and govern the emergence and behavior of cancer at all length scales. The study described in this manuscript focused on physical changes associated with metastasis. A controlled set of comparative studies of two cell lines that are extensively used as cell models of cancer metastasis and straddle the metastatic transition was undertaken by the PS-OC Network. The cell lines analyzed were the immortalized human breast epithelial cell line MCF-10A, representing a nontumorigenic state, and the human metastatic breast cell line MDA-MB-231, representing a malignant state. Distinguishing features of the adherent, non-transformed, MCF-10A cells are their lack of tumorigenicity in nude mice, lack of anchorage-independent growth, and dependence on growth factors 4. In contrast, MDA-MB-231 cells 5 form highly malignant, invasive tumors in vivo, are resistant to chemotherapy drugs such as paclitaxel, exhibit anchorage-independent growth, and grow independently of growth factors. Although MCF-10A cells have wild-type p53 and MDA-MB-231 cells have mutant p53, both cell lines are negative for the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) 6,7. To ensure that data generated across the multiple PS-OC laboratories could be integrated, culture guidelines, common culture reagents, and the two fully characterized, karyotyped cell lines were distributed to PS-OC laboratories. This minimized phenotypic and genotypic drift. After demonstration of growth uniformity, the
Integrative Biology, 2015
Focal heating of endothelial cells with a surface microelectrode induces secretion of vWF, expres... more Focal heating of endothelial cells with a surface microelectrode induces secretion of vWF, expression of P-selectin, and recession of cell–cell junctions to reveal a subendothelial matrix that supports platelet adhesion and aggregation.
In May 2002, he received his Bachelor's of Engineering from the Department of Chemical Engineerin... more In May 2002, he received his Bachelor's of Engineering from the Department of Chemical Engineering with distinction. Following the bachelor's degree, the author worked with a pharmaceuticals intermediate manufacturer in Vadodara as a process development engineer.
Blood
Fibrinolytic drugs such as recombinant tissue plasminogen activator (tPA) have been successfully ... more Fibrinolytic drugs such as recombinant tissue plasminogen activator (tPA) have been successfully used to reestablish blood flow in thrombotic strokes. However, they can also lead to intracranial hemorrhaging, must be used within hours of the onset of symptoms, and have limited efficacy in small vessel occlusions distal to major cerebral arteries. Alternatively, catheter-based thrombectomy devices can restore blood flow quickly but are invasive, limited to larger arteries, and can leave residual prothrombotic material on vessel walls. An approach that overcomes these limitations is the injection of individual superparamagnetic particles coated with fibrinolytic drugs that, upon application of a rotating magnetic field, self-assemble into microbots we call microwheels capable of translating to and directly attacking thrombi. We describe an approach that uses the vessel wall to propel in situ assembled microwheels via rolling.1 Driven by low-strength external magnetic fields (~10 mT), ...
Bulletin of the American Physical Society, 2017
Cellular and Molecular Bioengineering
Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The ty... more Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The type of thrombus that forms depends on the nature of the injury and hemodynamics. There are many models of intravascular thrombus formation whereby blood is exposed to prothrombotic molecules on a solid substrate. However, there are few models of extravascular thrombus formation whereby blood escapes into the extravascular space through a hole in the vessel wall. Here, we describe a microfluidic model of hemostasis that includes vascular, vessel wall, and extravascular compartments. Type I collagen and tissue factor, which support platelet adhesion and initiate coagulation, respectively, were adsorbed to the wall of the injury channel and act synergistically to yield a stable thrombus that stops blood loss into the extravascular compartment in~7.5 min. Inhibiting factor VIII to mimic hemophilia A results in an unstable thrombus that was unable to close the injury. Treatment with a P2Y12 antagonist to reduce platelet activation prolonged the closure time twofold compared to controls. Taken together, these data demonstrate a hemostatic model that is sensitive to both coagulation and platelet function and can be used to study coagulopathies and platelet dysfunction that result in excessive blood loss.
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Papers by Kuldeepsinh Rana