Research On The Acquisition Method Of Tissue Section Micro-region Samples Based On Glass Microneedle

Spatially resolved transcriptomics has become another research hotspot after single-cell transcriptomics,which obtains gene expression information as well as the original spatial location of sample.Spatial transcriptome technologies have become powerful means to study the spatial heterogeneity of biological tissues.Among current spatial transcriptome technologies based on microdissection,the most widely used is LCM,which utilizes laser to precisely cut ROI of tissue section and is compatible with FFPE tissue.In view of the present shortage of LCM system,such as the expensive price,relatively complicated procedure,limited sample collection rate and possible nucleic acid damage,an alternative method is urgently needed to acquire micro-region samples at specific sites on tissue section for meeting the sampling needs of low-throughput spatial transcriptomics research.On the basis of full investigation of microdissection technology and pre-experimental results,this paper designs and produces a glass microneedle-based device for acquiring microregion sample on tissue section.Subsequently,the sampling protocol is optimized and the applicable conditions and performance of the device are explored during use.The main contents in this paper are as follows:1.A microneedle-based acquisition device of tissue micro-region sample is developed.Glass microneedles are used as the microdissection tool.Once the tip of sharp needle contacts with the tissue section,the micro-region of tissue section in the circular area is cut off under pressure.The designed device mainly includes a motorized stage,a microneedle and its control module,an inverted fluorescence microscopic imaging module and a lateral microscopic observation module,which can realize the selection of ROI sample on tissue sections,microdissection and transfer.2.Tissue micro-sample acquisition scheme is optimized.Transcriptomics experiments deal with easily degradable m RNA,and the optimization of sampling protocol can better transcript quality.Here,the preparation parameters of microneedles with specific diameter are obtained,which increase the utilization rate of microneedles;the buffer slides made by PDMS film and plasma bonding is selected to prevent microneedles from breaking and improve sampling success rate;the means of sample transfer which is that microneedles suck a small amount of lysis before sampling and drain micro-sample immediately is determined to reduce the loss of nucleic acid.3.Highly efficient sampling of frozen sections was achieved,and the applicable conditions and performance of the device were explored.Sampling and sequencing analysis of frozen tissue sections verify the reliability of the device.Furthermore,the results show that the device is suitable for frozen tissue sections,its spatial resolution reaches the single-cell scale(10 μm),its collection time of a sample is 2 min,and its success rate of sample capture is as high as 91.7%.The self-designed glass microneedle-based acquisition device for micro-region tissue has improved performance in the continuous optimization of operation process.It can be applied to frozen tissue micro-samples acquisition under brightfield and fluorescence observation.In addition,it owns the advantages with single-cell scale resolution,high time efficiency,high success rate and low cost,which make it possible to become a potential device to the LCM system.The device becomes another efficient method for studying the spatiotemporal heterogeneity of tissue samples.