Category: Cellular Technologies
Single cell analysis (SCA) or tissue microdissection are prerequisite steps in current biomedical research. Important part of SCA is the investigation of single cell adhesion properties that is a key in substrate mediated cell behavior essential for understanding cellular properties in health and disease. For SCA, there are commercially available cell collection technologies including laser based and cell sorting instruments. There are also approaches for single cell adhesion force measurements including atomic force microscopy, optical tweezers, and micropipette/capillary aspiration. However, none of the existing instruments provide concurrent single cell acquisition and adhesion force measurement. Also, current microdissection platforms are typically sample specific, complex and expensive making integration within standard lab/clinical sample processing workflows difficult. On the other side of this spectrum is tissue microdissection from formalin fixed paraffin embedded (FFPE) tumor biopsies and archived tissue samples is one of the most desirable yet technically challenging applications. None of the existing microdissection approaches offer convenient and cost efficient approach for FFPE tissue microdissection. Here, using our vacuum impulse based cell and tissue acquisition technology (CTAS; UP8797644) we developed automated system for single cell adhesion assay and fixed FFPE tissue microdissection. For single cell experiments, adhesion strength for individual cells from SH-SY5Y, CHO, 3T3, neural progenitor cell cultures were measured using a sensor incorporated in the collection assembly. Collected single cells were dispensed automatically into individual wells for further interrogation. A new protocol for fixed tissue microdissection was developed using a novel heat activated alkaline based DNA extraction method to acquire region specific DNA from PFA-fixed and FFPE archived tissue sections ranging in thickness from 5 to 20 µm. Tissue dissociation was achieved using NaOH solution preloaded into disposable capillary units (DCUs; 1 to 5 µl) with a custom microscope stage heater. Developed instrument collects individual cells from any adherent cultures grown in standard cell culture dishes in as small as 15 nl volume, compatible with various downstream single cell analyses and next generation sequencing. Cell adhesion/detachment assay was performed based on the capillary aspiration techniques reported earlier. Collection of tissue regions of interests (ROIs) down to 20 µm in diameter was efficiently performed on both PFA and FFPE tissue sections within 30 seconds. A tissue dissociation step ensured compatibility of isolated DNA with PCR amplification. Both manual and automatic extraction and deposition algorithms in the instrument’s software were developed for acquisition of targeted single cells and ROIs. We have developed a universal platform for fresh frozen and fixed (FFPE) tissue microdissection, single cell acquisition, measurements of its adhesion strength and automatic deposition into the single wells for further molecular analysis or clonal expansion making it the most cost-efficient and versatile single cell analysis and tissue microdissection platform currently available.
Stanislav Karsten– President and CEO, NeuroInDx, Inc., Torrance, CA
President and CEO
Dr. Karsten is a President and CEO of NeuroIndx, inc. He is an author of the original cell and tissue acquisition concept and co-founder of NeuroInDx, Inc. Dr. Karsten started to work on methods development and optimization in Uppsala University in 1994, received his Ph.D. in Medical Genetics and Pathology from the same university in 2000 and continued his work in UCLA (Neurogenetics fellow). Prior to moving to NeuroInDx, Dr. Karsten served as a Chief of the Division of Neuroscience at Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Dr. Karsten served as a PI on multiple grants and has 8 patent applications and over 40 peer reviewed publications and chapters.