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How to generate tissue arrays for Xenium?

Question: How to generate tissue arrays for Xenium?

 

Answer:
Here we provide a summary of learnings for tissue array constructions. These tips and tricks have been developed by the Xenium sample preparation team and apply to the Xenium product family only. The documentation provided here is officially unsupported. 10x Genomics support will not be able to provide additional information outside what is stated in this article or provide replacement reagents should suboptimal data be obtained with these methods.

 

Tissue Arrays (TAs) are like mosaics, with different tissue samples embedded into a single block. TAs provide a holistic view of tissue architecture and cell types, enabling high-throughput analysis for large scale studies. This type of screening method can be beneficial because it can enable the simultaneous assessment of multiple tissue/donor samples. In addition, FFPE samples ensure the preservation of tissue architecture and cellular morphology, while offering long term sample stability. Unlike Tissue MicroArrays (TMA), which are assembled from small cylindrical cores and only represent a small snapshot of the overall tissue, microstrips offer greater spatial context. TAs allow for a broader assessment of a tissue composition and cellular interactions and can be advantageous for studying complex structures and heterogeneity within tissues.

 

Figure 1. Tissue Array vs. Tissue Microarray. Tissue array contains fewer tissues, but with large area.

Tissue Arrays allow for cost efficient screening of multiple tissue types for Xenium.

 

Figure 2. Example Tissue Array for a single Xenium slide containing seven different tissues that fit in the Xenium sample area.

 

Methods Overview: 

First, FFPE blocks containing tissue of interest are obtained and then region(s) of interest are identified. Tissue is released from the mold by gentle heating and regions of interest excised. The tissue is then arrayed and embedded.

 

Figure 3. Methods overview. (1) FFPE tissue blocks are obtained. In this example, seven blocks are selected (2) Tissue is QC’d and tissue regions are identified. (3) Tissue is arranged in a single mold that fits the Xenium Sample Area.

 

Detailed Methods: 

Reagents and equipment: 

*reagent swaps are low risk; reagents and equipment used at 10x linked above.

 

Getting started:

  • Select High-Quality Samples. Avoid thick, hard samples like bone or calcified tissues that could damage adjacent tissue strips during sectioning. Also, avoid poorly processed samples. Tissue QC (with H&E) is critical for sample selection. Also ensure FFPE tissues have been optimally fixed.
  • Consider Floatation Times. Different tissues have varying floatation times. Ensure tissues with significantly different times (e.g., skin vs. spleen) are not placed together.
  • Allow for Tissue Expansion 
    • Keep in mind that samples expand once they're floating. Avoid over-packing to prevent loss of samples from the capture area.

Design Considerations:

  • The Tissue strip dimensions will vary depending on the TA design. The ideal dimension per tissue strip is 8mm x 2mm to comfortably fit seven tissues within the Xenium capture area. Uniform sample thickness in a single TA ensures minimal empty regions and maximizes block longevity for sectioning.
  • Max width: 8mm per sample.

Selecting the tissue region:

  1. Obtain single FFPE samples and label them for identification).
    • Tip: Ensure proper labeling to maintain sample traceability and organization throughout the TA construction process.
  2. Examine each sample under a microscope and identify the tissue region for further analysis.
    • Tip: Using a staining method of choice, identify regions with pathological features, unique cell populations, or areas demonstrating relevant biomarkers for experimental purpose.
    • Avoid selecting regions of poor tissue quality, details can be found in Xenium FFPE Tissue Preparation Guide (CG000578).
  3. Mark the region of interest (ROI) on the surface of the FFPE sample using a marker.

 

Figure 4. Tissue region selection. (1) Blocks are obtained and tissue orientation is noted. (2) Blocks are QC’d and region(s) are identified. (3) Regions are marked directly on the block with a pen.

 

Melting and Isolating Tissue:

Here are an outline of steps for melting and isolating tissue prior to tissue array construction. During this process, it is critical to avoid excessive heat. This is important to preserve tissue morphology and molecular components (e.g., nucleic acids).

  1. Preheat the Epredia HistoStar embedding station and heating chamber to the specified temperature for paraffin wax melting (approximately 58-60°C).
  2. Carefully place the FFPE sample(s) in the chamber and gently apply heat to melt the paraffin, ensuring minimal degradation of nucleic acids.
    • Tip: Monitor the melting process to prevent overheating or damage to the sample.
  3. After the paraffin has liquefied, isolate and transfer the tissue sample onto a flat surface, free from any residual paraffin.
    • Tip: To expedite the paraffin melting process, consider using a single-edge razor blade to delicately remove excess paraffin and detach the sample from the tissue cassette. 
  4. Keep track of the sample's orientation & location. Proceed with repeating the melting & collection process for additional samples to be re-embedded.
    • Tip: Once all tissue samples have been freed from paraffin, arrange them in the mold to assess their size and dimensions. Trim as necessary to ensure a proper fit for all tissues within the mold for downstream processing.

Figure 5. Tissue samples prior to remolding. (1) Tissue samples released from paraffin by melting. (2) Tissue samples arranged in mold to assess fit.

 

Embedding of Samples into a FFPE Block:

Here is an outline of steps for embedding the isolated tissue in to a single FFPE block. As a best practice, it is good to use extra forceps. Having spare forceps on hand helps during the construction process. Switch them when paraffin solidifies, ensuring precision.

  1. Prepare the Metal base mold for tissue embedding within the Epredia HistoStar embedding station.
  2. Preheat the embedding station to the recommended temperature for embedding paraffin-embedded tissue (approximately 58-60°C).
  3. Create a base layer in the metal mold by adding a small amount of melted paraffin wax.
  4. Swiftly transfer the first collected melted tissue sample with forceps onto the paraffin base layer within the mold. Sit tissue flush against the mold.
  5. Solidify the paraffin rapidly, either by using the Epredia HistoStar cooling function or by placing the mold on a cooling plate.
  6. Repeat steps 3-5 for each additional melted tissue sample, layering them sequentially within the mold.
  7. Ensure that each layer of paraffin solidifies before adding the next sample to maintain separation between tissues.
  8. Once the block is fully solidified with all tissues, proceed with microtomy and slide preparation.
    • Tip: Adjust the microtome to proper sectioning plane. Tissue array constructs may lead to loss of ~20-50 um worth of sections until all tissue strips are exposed.

Panel selection: 

Assay performance is dependent on both sample quality, but also tissue to panel match. It will be important to select a panel with good representation of genes across the tissue types present in the array. The multi panels are designed to accommodate multiple human or mouse tissue types, respectively. Prior to performing the first Xenium In Situ Gene Expression experiment on a tissue array, it is recommended to compare the genes in the panel across the expected transcriptome of the array tissues. Alternatively, an add-on or standalone custom panel can be designed specifically to match a custom array.

 

Drawbacks: 

  1. Costly & Resource-Intensive: FFPE Tissue Array construction can be quite expensive (pending array layout) - it involves sample procurement ($$$), specialized equipment ($$$), labor/time from trained personnel ($$), and various consumables ($). 
  2. Sample Thickness Limitations: Facing and sectioning the TA block may lead to pockets of exhaustion within the block. Longevity is a key concern, especially if all samples within the array have varying thickness.
  3. Tissue Heterogeneity: Snippets or smaller chunks to assemble an array may not fully capture the heterogeneity present within the original/larger donor tissue. Variations in cellular composition or morphology may be underrepresented. 
  4. Potential Tissue Damage: During the construction or sectioning of FFPE Tissue Arrays, there are risks that the workflow may damage or compromise the tissue. Example is a nicked blade that may run across all arrays, or heat induced damaging during the re-embedding, which may lead to decreased assay performance. 
  5. Higher gDNA: Tissue arrays general have higher gDNA relative to control blocks. Differences in gDNA in TAs likely related to heating → damages tissue.

Conclusions: 

  • Constructing FFPE Tissue Arrays is a meticulous process requiring various considerations in order to successfully generate, and obtain high quality data. 
  • Factors such as: dimensions, layout, tissue grouping (based on float time), and preventing tissue damage during construction are paramount for high-quality arrays.
  • Optimization Matters: Depending on research objectives and tissue characteristics, additional optimization may be necessary.

Figure 6. Example Tissue Array. Eight different tissues are molded in one block starting with breast (1, left) and ending with kidney (8, right).

 

Product: Xenium In Situ Gene Expression