Acoustofluidic Medium Exchange for Preparation of Electrocompetent Bacteria Using Channel Wall Trapping

Transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturize this step, we developed an acoustofluidic device capable of trapping the bacterium <i>Escherichia coli </i>non-invasively for subsequent exchange of medium, which is challenging in acoustofludic devices due to detrimental acoustic streaming effects. <br>With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilized for trapping of bacteria. Our novel design robustly traps <i>Escherichia coli</i> at a flow rate of 10 µL minute^-1 and has a cell recovery performance of 47 ± 3 % after washing the trapped cells.<br>To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8∙10⁵ CFU per µg of plasmid DNA. Our device is a viable low-volume alternative to centrifugation-based methods and opens the door for miniaturization of a plethora of microbiological and molecular engineering protocols.<br>

Acoustofluidic Medium Exchange for Preparation of Electrocompetent Bacteria Using Channel Wall Trapping

Transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturize this step, we developed an acoustofluidic device capable of trapping the bacterium <i>Escherichia coli </i>non-invasively for subsequent exchange of medium, which is challenging in acoustofludic devices due to detrimental acoustic streaming effects. <br>With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilized for trapping of bacteria. Our novel design robustly traps <i>Escherichia coli</i> at a flow rate of 10 µL minute^-1 and has a cell recovery performance of 47 ± 3 % after washing the trapped cells.<br>To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8∙10⁵ CFU per µg of plasmid DNA. Our device is a viable low-volume alternative to centrifugation-based methods and opens the door for miniaturization of a plethora of microbiological and molecular engineering protocols.<br>

Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping

Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. We present an acoustofluidic chip, capable of automated medium exchange of bacteria.

Dielectrophoretic medium exchange around droplets for on-chip fabrication of Layer-by-Layer microcapsules

Continuous medium exchange within a microchannel represents a highly sought-after technique in functionalizing micro-objects with coating layers, enabling a myriad of applications ranging from biomedical engineering to materials science. Herein,...

Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture

ABSTRACTMalaria infection by Plasmodium falciparum continues to afflict millions of people worldwide, with transmission being dependent upon mosquito ingestion of the parasite gametocyte stage. These sexually committed stages develop from the asexual stages, yet the factors behind this transition are not completely understood. Here, we found that lactic acid increases gametocyte quantity and quality in P. falciparum culture. Low-passage-number NF54 parasites exposed to 8.2 mM lactic acid for various times were monitored using blood film gametocyte counts and RNA analysis throughout 2 weeks of gametocyte development in vitro for a total of 5 biological cohorts. We found that daily continuous medium exchange and 8.2 mM lactic acid supplementation increased gametocytemia approximately 2- to 6-fold relative to controls after 5 days. In membrane feeding mosquito infection experiments, we found that gametocytes continuously exposed to 8.2 mM lactic acid supplementations were more infectious to Anopheles stephensi mosquitoes, essentially doubling prevalence of infected midguts and oocyst density. Supplementation on days 9 to 16 did not increase the quantity of gametocytes but did increase quality, as measured by oocyst density, by 2.4-fold. Lactic acid did not impact asexual growth, as measured by blood film counts and luciferase quantification, as well as radioactive hypoxanthine incorporation assays. These data indicate a novel role for lactic acid in sexual development of the parasite.

A Facile Approach for Rapid Prototyping of Microneedle Molds, Microwells and Micro-Through-Holes in Various Substrate Materials Using CO2 Laser Drilling

CO2 laser manufacturing has served as an enabling and reliable tool for rapid and cost-effective microfabrication over the past few decades. While a wide range of industrial and biological applications have been studied, the choice of materials fabricated across various laser parameters and systems is often confounded by their complex combinations. We herein presented a unified procedure performed using percussion CO2 laser drilling with a range of laser parameters, substrate materials and various generated microstructures, enabling a variety of downstream tissue/cellular-based applications. Emphasis is placed on delineating the laser drilling effect on different biocompatible materials and proof-of-concept utilities. First, a polydimethylsiloxane (PDMS) microneedle (MN) array mold is fabricated to generate dissolvable polyvinylpyrrolidone/polyvinyl alcohol (PVP/PVA) MNs for transdermal drug delivery. Second, polystyrene (PS) microwells are optimized in a compact array for the formation of size-controlled multicellular tumor spheroids (MCTSs). Third, coverglass is perforated to form a microaperture that can be used to trap/position cells/spheroids. Fourth, the creation of through-holes in PS is validated as an accessible method to create channels that facilitate medium exchange in hanging drop arrays and as a conducive tool for the growth and drug screenings of MCTSs.

Sirolimus-Eluting Electrospun-Produced Matrices as Coatings for Vascular Stents: Dependence of Drug Release on Matrix Structure and Composition of the External Environment

Although a number of drug-eluting coatings for vascular stents (VSs) have been developed and are in commercial use, more efficient stent coatings and drug delivery systems are needed. Sirolimus (SRL) is a clinically important drug with antiproliferative and immunosuppressive activities that is widely used for coating stents. Here, we characterized SRL-enriched matrices, intended for coating vascular stents, that were produced by electrospinning (ES) on a drum collector from a solution of polycaprolactone (PCL) and human serum albumin (HSA), 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), dimethyl sulfoxide (DMSO), and SRL. The release of tritium-labeled SRL (3H-SRL) from matrices in phosphate-buffered saline (PBS) or human blood plasma (BP) was studied. The introduction of DMSO in the ES blend decreased SRL release. The use of BP significantly accelerated SRL release through binding with serum biomolecules. The exchange of PBS or BP after every time point also increased SRL release. The maximum SRL release in BP was observed at 3 days. The matrices produced from the ES solution with DMSO and HSA released no more than 80% SRL after 27 days in BP, even under medium exchange conditions. Therefore, PCL-based matrices containing HSA, SRL, and DMSO can be used for coating VSs with prolonged SRL delivery.

CXCL-13 Regulates Resistance to 5-Fluorouracil in Colorectal Cancer

Purpose5-Fluorouracil (5-Fu) is used as a conventional chemotherapy drug in chemotherapy for patients with advanced colorectal cancer, but many patients still suffer from treatment failure due to 5-Fu resistance. Emerging observations revealed the important role of chemokine (C-X-C motif) ligand 13 (CXCL-13) in tumor microenvironment and its relationship with prognosis in patients with colorectal cancer. This study is designed to reveal the important role of CXCL-13 in causing colorectal cancer resistance to 5-Fu.Materials and MethodsCXCL-13 levels of patient's serum or cell culture supernatants were measured separately by enzyme-linked immunosorbent assay. In cell assays, cell viability is detected by Cell Counting Kit-8. Therefore, the recombinant human CXCL-13 was used to simulate its high expression in cells while its antibody and siRNA were used to reduce CXCL-13 expression in cells.ResultsIn this study, we demonstrated that CXCL-13 is associated with 5-Fu resistance by culture medium exchange experiments and cytokine arrays of colorectal cancer resistant and nonresistant cells. Clinical studies showed that CXCL-13 is highly expressed in the serum of 5-Fu–resistant patients. High levels of serum CXCL-13 also predict a worse clinical outcome. The addition of recombinant CXCL-13 cytokine resulted in 5-Fu resistance, while its antibody overcame 5-Fu resistance, and knockdown of CXCL-13 expression by siRNA also reduced 5-Fu resistance, which can be saved by added recombination CXCL-13.ConclusionThese results not only identify a CXCL-13 mediated 5-Fu resistance mechanism but also provide a novel target for 5-Fu–resistant colorectal cancer in prevention and treatment strategies.