Dr. Maggie Witek ( mwitek@ku.edu)
Dr. Mengjia Hu ( m367h922@kumc.edu)
1020 Wahl Hall East, KUMC
The liquid biopsy core can perform the isolation of circulating markers such as rare cells (for example, circulating tumor cells, CTCs), cell free molecules (such as circulating cell free DNA), and extracellular vesicles (EV) in highly efficient and reproducible ways with full automation. The Core can isolate LB markers from a variety of clinical samples including blood, urine, sweat, saliva, and cerebrospinal fluid as well as others. Following the isolation, the amount (number of cells or EV particles or the mass of cell free molecules) of enriched LB marker can be counted and the marker subjected to a variety of molecular assays, such as droplet digital PCR, RT-qPCR, Western blotting, and Next Generation Sequencing. However, the isolate can be returned to the user for molecular processing in his/her own laboratory. A robotic platform can accept samples in standard tubes, such as blood EDTA tubes, and fully automate the isolation using unique technology specific to the Core. Blood samples can be seeded with a stabilizer cocktail to allow shipment of samples at room temperature to the Core (stability for up to 72 h post-draw).
Due to the robotic nature of the isolation process, the results are highly reproducible and the Core can perform 36 isolations per day. The Core has a specially designed microfluidic chip for optimal performance in terms of recovery and purity for the isolation of rare cells, cell free molecules, and EVs. The same robot can process a clinical sample for any of the LB markers by selecting the appropriate microfluidic chip.
Liquid handling robot for operating microfluidic chips specifically designed for the high efficiency isolation of LB markers. The chips are operated by a pair of pipet tips that push/pull the clinical sample through the chip. The chips are made from a plastic via injection molding and thus, high volumes of chips can be produced at low cost to support any clinical trial. The robot can operate 8 chips simultaneously. Specific chips are designed for isolating biological cells, cell free molecules and extracellular vesicles.
We can process samples for internal and external users (academic and private). RATES for isolation of Rare Cells, EVs, or cfDNA are $75 (KU-L and KUMC users), $145 for external academic clients, and $225 for private sector clients. If you require enumeration of the isolated markers, additional charges will incur. This can be discussed on a case-per-case basis with the cost dependent on the marker to enumerate (CTCs versus EVs) and the assays that are performed for the enumeration. We can determine cfDNA content as well from the isolate.
Other equipment available to the Core:
We use a plastic chip to affinity isolate the biological cells of choice by the user. The plastic chip can be tailored for any antibody or other affinity agent, such as aptamers to target the necessary cells. The chip has been demonstrated to efficiently isolate CTCs, circulating leukemia cells, circulating multiple myeloma cells, and leukocyte subsets.
Rare cell affinity isolation chip. The chip is made from a plastic via injection molding and consists of a series of sinusoidally-shaped channels. Each channel contains affinity agents that can be tailored to fit the user’s application. This chip can be operated by the robotic platform.
The Core uses a plastic chip consisting of a high density array of micropillars (each chip has 1.5M pillars) that can be activated to allow for the covalent attachment of affinity agents to each pillar. The high surface area of the chip allows for selecting >1011 particles, and release the EVs in a total volume of 22.4 µ L. The chip is manufactured via injection molding in a high production mode. The chip can recover >80% of the EVs at a volume flow are ~25 µ L/min. The chip can be operated on the fluid handling robot.
EV affinity isolation chip. The chip is made from a plastic, in this case cyclic olefin copolymer (COC), that can be UV/O3 activated to allow for direct antibody attachment. Each pillar contains surface immobilized affinity agents (any affinity agent can be used to suit the application need). The chip can be operated by the liquid handling robot. Because the chip is made via injection molding, the cost per assay is low and any clinical study can be supported.
We have developed an inexpensive microfluidic chip that can be used for the solid-phase extraction of DNA (cfDNA, plasmid DNA, genomic DNA) with high efficiency and using a very simple procedure. The chip is made from a plastic that can be injection molded to produce the chip at low cost and in high numbers to support any clinical study. When compared to existing solid-phase extraction techniques (Qiagen, Norgen), it exceeds their recovery values across a large DNA size range (recovery >85%). In addition, the solid-phase extraction chip can be used to extract total RNA as well. The chip can be operated by our fluid handling robot and process 16 samples at any one time and with full automation. Here are the salient features of this solid-phase extraction technique: