"Open-top" microfluidic device for in vitro three-dimensional capillary beds.

Human neural stem/progenitor cells (hNSPCs) have the potential to widen the current narrow treatment window for stroke as they improve functional recovery in rodent stroke models when transplanted weeks after stroke. One aspect of the hNSPC-induced functional recovery is increased angiogenesis and neovascularization in the peri-infarct region. Our lab created a human cell in vitro model of vessel formation by seeding hNSPCs and human endothelial progenitor cells (hEPCs) in a 3D scaffold composed of salmon fibrinogen, laminin, and hyaluronic acid that mimics brain tissue properties. Using our in vitro neurovascular model, we tested the hypothesis that hNSPC-secreted material plays a role in the stimulation of vessel formation. Our RNA-Seq data show that hNSPCs express high levels of secreted pro-angiogenic proteins, such as growth factors, matrix molecules, and cytokines, but hNSPCs might also impact vessel formation by secretion of extracellular vesicles or cell-contact mediated mechanisms. In order to determine the effect of hNSPC-secreted material on vessel formation, mCherry-labeled hEPCs were seeded in 3D scaffolds alone, with CellTracker Green-labeled hNSPCs, or with hNSPC-conditioned media containing hNSPC-secreted soluble factors and extracellular vesicles, such as exosomes. Vessel formation was quantified using AngioTool to determine total vessel length, number of branch points, and vessel percentage area. We found an increase in vessel formation in the presence of hNSPCs and hNSPC-conditioned media compared to hEPCs alone. In conclusion, material secreted by hNSPCs can recapitulate the increase in vessel formation induced by hNSPCs themselves. In future studies, we will determine whether hNSPC-derived exosomes are important for promoting vessel formation as they have therapeutic potential without the limitations of cell therapy.

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.

Polymeric substrates have significant advantages over silicon and glass for use in microfluidics. However, before polymer microfluidic devices can be mass produced, it must be shown that the manufacturing method used to create these devices is robust and repeatable. For this paper, a polymer manufacturing process, hot embossing, was used to produce microsized features in polymethylmethacrylate (PMMA) chips. A design of experiments that varied two factors during the hot embossing process (temperature and pressure), was conducted to determine the robustness of hot embossing microsized channels in PMMA. The channel height and width were measured at three sites on each chip, and the results were analyzed in two ways: response surface modeling (RSM) and nested variance analysis. For the RSM analysis, two separate ANOVA tests and regressions were performed on both channel width and channel height to obtain the response surface models between temperature, pressure and the channel width and height. Furthermore, the variance of channel width and height at each design point was determined and then two ANOVA tests and two separate regressions were performed to obtain the response surface models between temperature, pressure and the variance of channel height and channel width. This analysis was used to determine if hot embossing microfluidic devices is a robust process capable of producing quality parts at different operating conditions. The nested variance analysis was used to determine the primary source of the variation in channel height and width. For the nested variance analysis, two separate calculations were performed in order to determine whether the variance of channel width and height is mostly caused by within-chip variance or chip-to-chip variance. The analysis showed that the channel widths and heights were statistically equal across the four different operating points used (the low-temperature, low-pressure point was omitted). The variance of channel width and the variance of channel height remained constant in the desired operating region. Based on this analysis, it was concluded that hot embossing is a robust process for features on the order of 50 μm. Furthermore, the nested variance analysis showed that the variance of channel width and height is mostly caused by site-to-site measurements on a chip rather than between-chip variance. Therefore, it was determined that hot embossing microfluidic devices are repeatable and consistent from chip-to-chip.

We present a simple, fast, and inexpensive new printing-based fabrication process for flexible and wearable microfluidic channels and devices. Microfluidic devices are fabricated on textiles (fabric) for applications in clothing-based wearable microfluidic sensors and systems. The wearable and flexible microfluidic devices are comprised of water-insoluable screen-printable plastisol polymer. Sheets of paper are used as sacrificial substrates for multiple layers of polymer on the fabric’s surface. Microfluidic devices can be made within a short time using simple processes and inexpensive equipment that includes a laser cutter and a thermal laminator. The fabrication process is characterized to demonstrate control of microfluidic channel thickness and width. Film thickness smaller than 100 micrometers and lateral dimensions smaller than 150 micrometers are demonstrated. A flexible microfluidic mixer is also developed on fabric and successfully tested on both flat and curved surfaces at volumetric flow rates ranging from 5.5–46 ml min−1.

In this study, we propose a novel method to generate a capillary pressure-driven flow in a microchannel with a hydrophobic surface. The microfluidic device has a wide channel in which a hydrogel pillar array is embedded. The hydrogel pillar array was formed in the microchannel by a photopolymerization process. The flow rate due to a capillary action was strongly dependent on the distance between the pillars. Moreover, our capillary pumping with a hydrogel pillar array sustained the flow for more than 5 min with a limited sample volume. Our microfluidic device provides two advantages: (1) the modification of the polymer surface to make it hydrophilic is not required and (2) the conventional polymer molding technique can be applied to produce microfluidic devices, instead of the precision molding technique. The results indicate the possible fabrication of various microfluidic chip devices that can be easily implemented in point-of-care diagnostics.

Transplantable ready-made microvessels have therapeutic potential for tissue regeneration and cell replacement therapy. Inspired by the natural rapid angiogenic sprouting of microvessels in vivo, engineered injectable 3D microvessel networks are created using thermoplastic elastomer (TPE) microfluidic devices. The TPE material used here is flexible, optically transparent, and can be robustly yet reversibly bonded to a variety of plastic substrates, making it a versatile choice for microfluidic device fabrication because it overcomes the weak self-adhesion properties and limited manufacturing options of poly(dimethylsiloxane) (PDMS). By leveraging the reversible bonding characteristics of TPE material templates, we present their utility as an organ-on-a-chip platform for forming and handling microvessel networks, and demonstrate their potential for animal-free tissue generation and transplantation in clinical applications. We first show that TPE-based devices have nearly 6-fold higher bonding strength during the cell culture step compared to PDMS-based devices while simultaneously maintaining a full reversible bond to (PS) culture plates, which are widely used for biological cell studies. We also demonstrate the successful generation of perfusable and interconnected 3D microvessel networks using TPE-PS microfluidic devices on both single and multi-vessel loading platforms. Importantly, after removing the TPE slab, microvessel networks remain intact on the PS substrate without any structural damage and can be effectively harvested following gel digestion. The TPE-based organ-on-a-chip platform offers substantial advantages by facilitating the harvesting procedure and maintaining the integrity of microfluidic-engineered microvessels for transplant. To the best of our knowledge, our TPE-based reversible bonding approach marks the first confirmation of successful retrieval of organ-specific vessel segments from the reversibly-bonded TPE microfluidic platform. We anticipate that the method will find applications in organ-on-a-chip and microphysiological system research, particularly in tissue analysis and vessel engraftment, where flexible and reversible bonding can be utilized.

Introduction: Glioblastoma (GBM) is one of the most aggressive malignant brain tumors. Many studies have reported that the tumor microenvironment (TME) contributes greatly to invasion and drug resistance of GBM. Our lab has previously described a tri-culture model using methacrylamide-functionalized gelatin (GelMA) with brain microvascular endothelial cells (BMVEC), human normal astrocytes (NHA), and human brain vascular pericyte (HBVP) recapitulated the in vivo brain microenvironment. However, phenotypic drug response was only observed under supraphysiological dosage. To evaluate therapeutic response more precisely, we decided to combine the Tri-culture system with the IdenTx3 microfluidic device (AIM Biotech). By separating the immature perivascular network from the GBM spheroids, we are hoping to generate a model that can more realistically mimic the TME. While many studies have used cancer spheroid in GBM studies, little is known about the impact of spheroids size. Since spheroid size correlates with nutrient and oxygen gradients, we are also curious to find out whether an increase in spheroid size increases the necrotic population and affects drug response and invasion. Materials: Wild-type (WT) and TMZ resistant 42MGBA cell lines were seeded at different cell numbers to generate spheroids of different sizes. Spheroids were dissociated for flow analysis to quantify the necrotic population. In the invasion assay, spheroids were encapsulated in GelMA hydrogel with or without the addition of perivascular cells including BMVECs, HBVP, and NHA (3:1:1). Vasculature formation inside the chip was initiated 5 days prior to the introduction of GBM spheroids. Results: 42MGBA-WT and 42MGBA-TMZres spheroids demonstrated phenotypical and proliferative differences. Spheroids sizes showed a direct relationship with necrotic cell numbers. Small spheroids (1000 cells) and large spheroids (10,000 cells) were encapsulated inside the hydrogel and subjected to different treatments of temozolomide. A difference in invasion area and proliferation was found when subjected to bulk or metronomic dosages. The two 42MGBA cell lines displayed an increase in invading area when co-cultured with perivascular cells. This result coincided with our previous finding that perivascular network increases GBM proliferation and invasion in GBM6 spheroid (Ngo et al, 2022). In addition, a different invasion behavior was observed between the 42MGBA-WT and 42MGBA-TMZres spheroids when introduced inside the microfluidic device. Surprisingly, the wildtype showed a larger invading area compared to the TMZ resistant. Conclusion: We demonstrated a direct relationship between size and necrotic population in the 42MGBA spheroids. We also showed that bulk or metronomic treatment exert different effects on the cancer spheroids. Finally, we have verified that TME is critical to invasion and drug resistance. Citation Format: Sheridan Fok, Yoanna Ivanova, Victoria Kriuchkovskaia, Brendan Harley. Investigating size-dependent invasion behaviors and therapeutic responses of the 42MGBA glioblastoma spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 238.

High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

In 2012, more than half a million people have been projected to die of cancer in the United States of America alone. In spite of the best efforts by the research community, following the declaration of the war on cancer 40 years back, cancer incidents and death rates have declined marginally. These facts vividly show that cancer research needs a shift in the paradigm, and in this respect, microfluidics is expected to provide the next generation tools for the oncologists. In recent years, a significant volume of literature has appeared to demonstrate that microfluidic strategies could be used to throw new light on cancer development, detection, diagnosis, and treatment. These developments take advantage of the fact that at the operational scale of a typical microfluidic system, researchers are able to address a single biological cell, even a portion of it; generate complex and stable gradient of chemical compounds; assay the activities of several therapeutic agents simultaneously, using a very small volume of the reagents and biological samples; separate a specific single cell from a population with a million others; and above all, create a confined microenvironment that mimics the physiological milieu. The sheer diversity in design and functionalities of these microfluidic systems further illustrates the enormous scope for researchers, who are willing to apply microfluidics in cancer research. Nevertheless, many corners of cancer remain unexplored, and new research developments are always in progress. I feel extremely honored to edit this special topics section entitled Microfluidics in Cancer Research, and I am thankful to Dr. Leslie Yeo (Editor of Biomicrofluidics) for giving me the opportunity to do so. In this section, we have an assortment of 11 papers, which cover both the fundamental and applied aspects of this stimulating research domain.

Microfluidic devices make precisely controlled processing of substances possible on a microliter level. The advantage is that, due to the small sizes, the driving forces for mass and heat transfer are high. The surface to volume ratios are also high, which can benefit many surface oriented processes. In addition, because of their small volumes, microfluidic devices reduce reagent consumption and risk of failure compared to larger counterparts. Furthermore, the parallelization of such devices can increase productivity while maintaining their characteristics. Overall, these advantageous properties give many opportunities for reaction and separation processes. Although researchers have intensively studied microfluidics for analytical and sensory applications, microfluidics for preparative processes is still in its infancy. This thesis research involved exploring these processes for biocatalysis and bio-separations with microfluidic devices. The purpose of this thesis was to yield a better understanding of microfluidics for the preparative processes and larger-scale production. We therefore addressed subjects including microfluidic parallelization, membrane separation, biocatalysis, and design. The presented research is useful for further developing innovative process intensification by means of microfluidic devices. Parallelization of microfluidic devices can facilitate the generation of more data or product in less time. In Chapter 2, we present a proof of concept of such a parallelization for obtaining information on reaction and separation kinetics. We assembled different microfluidic contactors into a single device in order to perform distinct experiments simultaneously. The concept of the parallelization was based on the decoupling of pressure drop from residence time. We demonstrated this by microfluidic membrane separations and determination of membrane properties. The reported device enabled a three times higher throughput compared to devices with a single separation region. Processes such as chromatographic separation and nanofiltration can remove low molecular weight sugars from liquid mixtures of oligosaccharides. In Chapter 3, we present a novel separation process based on the concept of mass diffusion. Differences between diffusivities of the components drive such a separation, while membranes, in particular nanofiltration membranes, can enhance the separation. We demonstrate this by the use of a membrane microfluidic device for the separation of small molecular weight components. Our results show that mass diffusion separation in liquids is a feasible concept. With optimized microchannel and membrane dimensions, the presented separation process might compete with currently available separation technologies. For diffusion-based processes, such as mass diffusion separation shown in Chapter 3, small diffusion distances – and thus thinner membranes – can reduce diffusion times significantly. In Chapter 4, we used a microfluidic contactor to contact liquid streams via such extremely thin membranes. We show that the presented concept can be useful for diffusion-based pre-concentration or downstream processes such as fractionation and enrichment. Our results indicate that also this method can yield a feasible process. Moreover, the technology is generally applicable to any diffusing component – regardless of its absolute diffusivity or concentration. Fast mass transfer and low reagent consumption have made enzyme microreactors popular research tools. In Chapter 5, we used such a microreactor to study the effect of diffusion on enzyme activity. We found that the Michaelis-Menten kinetic parameters were similar at the microscale and bench scale. Our results show that with residence times below a few seconds, diffusion effects limited the reaction rate and therefore reduced the conversion per volume of enzyme microreactor. The critical residence time where this limitation occurred increased quadratically with channel width, increased with enzyme concentration, and decreased with substrate concentration. We concluded that in order to use an enzyme microreactor efficiently, such effects should be taken into account. Many parameters such as the enzyme properties, operating conditions, and dimensions of the microreactor determine to what extent mass transfer restrictions affect the reaction rate and the productivity. The use of microchannels can indeed shorten the characteristic mass transfer time, as shown in Chapter 5, but may also affect the productivity of the microreactor. Chapter 6 provides the correlations between these parameters for coflow enzyme microreactors obeying Michaelis-Menten kinetics. These correlations outline the design space based on reduced mass transfer restrictions and maximum productivity respectively. The methodology that yields the design space provides a generic hands-on approach to optimally design coflow enzyme microreactors. Microfluidics involves the exploitation of the phenomena that manifest themselves on microscale. This thesis shows that microscale applications can indeed offer unprecedented benefits. The discussion in Chapter 7 summarizes and reflects on the previous parts of this thesis. We conclude that it is important to explore and exploit other characteristics of continuous production in microfluidic devices beyond mass transfer effects in order to develop novel processes. In addition, we stress the importance of adoption of microfluidics, and show which determinants are involved in this. Knowledge of these determinants is of utmost importance to reduce skepticism towards and stimulate the adoption of microfluidics by industry.

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction.

Assessment of Red Blood Cell-Mediated Microvascular Occlusion in Sickle Cell Disease By a Novel Electrical Impedance-Based Microfluidic Device

Literature shows that EPCs contribute to increased collateral vessel formation following vaso-occlusion. However diabetes reduces EPC number and reduces collateral vessel formation. We cultured human EPCs and exposed them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to 360mg%, HG) glucose, which resulted in significant EPC death within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC reduction in hyperglycemia is secondary to up-regulation of P53. We obtained mouse EPCs from P53 null mice and observed that p53 KO EPCs are more resistant to cell death in HG. P53 null EPCs evolved into mature mouse EC (MEC) and retained endothelial properties. Next, we used Lenti and Adenovirus mediated si-RNA methods to silence P53 (long and short term respectively) in human EPCs and P53 silenced EPc showed better survival in HG. We developed femoral artery occlusion model to mimic peripheral vascular disease in diabetic animals. We used streptozotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null mouse EPCs intra-muscularly around the femoral occlusion in these mouse models (n=7 in each group) and counted CD31+ve number of capillaries in a 20x microscopic field. Highest number was noted in the group that received P53null EPCs. We also quantified increased vessel formation by laser doppler assay of the ischemic and contralateral hind limbs upto 2 weeks and conducted qRT-PCR and western blot of key endothelial genes such as eNOS, p-eNOS, VEGF-A, PECAM-1 and vWF from both quadriceps in all groups of mice. Summary: Transplantation of P53 null EPCs in hyperglycemic mice with femoral occlusion demonstrate better collateral vessel formation post femoral artery occlusion setting compared to WT-EPC transplanted group. Preliminary results from transplantation of p53 silenced human CD34+ cells also indicate increased vessel formation compared to non p53 silenced hCD34+ cell in NOD-SCID diabetic mice. Conclusion: This finding indicates towards possible therapeutic benefit in transplanting P53 silenced human EPC or CD34+ cells to prevent peripheral vascular disease in patients with diabetes.

Literature shows that EPCs contribute to increased collateral vessel formation following vaso-occlusion. However diabetes is associated with poor collateral vessel formation. We cultured human EPCs (defined as CD34+ cells) and exposed them to 5.5 mM (equivalent to 99 mg%, normal glucose, NG) and 20mM (equivalent to 360mg%, high glucose, HG) glucose, which resulted in significant EPC apoptosis within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC apoptosis in HG is secondary to up-regulation of P53 and silencing or knocking out P53 may help neovascularization. We obtained mouse EPCs from P53 KO mice and observed that p53 null EPCs are more resistant to cell death in HG while retaining expression of essential endothelial genes. Next, we used Lenti and Adenovirus mediated siRNA methods to silence P53 in human EPCs (hEPCs) and P53 silenced hEPC showed better survival in HG. We developed unilateral femoral artery occlusion model to mimic peripheral vascular disease in streptozotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null mEPCs intra-muscularly around the femoral occlusion (n=10 in each group) and counted number of capillaries in a 20x microscopic field. We also quantified increased vessel formation by laser doppler assay of the ischemic and contralateral hind limbs upto 2 weeks, post EPC transplantation and conducted qRT-PCR and western blot of key endothelial genes such as eNOS, p-eNOS, VEGF-A, PECAM-1 and vWF from both quadriceps in all groups of mice. Results: The group that received P53null EPCs had the most favorable outcome. Preliminary results from transplantation of lenti p53 silenced human CD34+ cells also indicate increased vessel formation compared to non p53 silenced hCD34+ cell in NOD-SCID diabetic mice. Summary: Transplantation of P53 null EPCs in presence of hyperglycemia help improve collateral vessel formation post femoral artery occlusion compared to WT-EPC transplanted group. Conclusion: Our results indicate towards positive role of apoptosis resistant CD34+ cells in diabetic PVD by increasing collateral formation with significant clinical implications.

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.