Question: Can cells be adhered Xenium slides to obtain gene expression data?
Answer: Below is a summary of our learnings and considerations from working with cultured HEK293T cells on the Xenium platform. Methods for PBMC adherence can be found in: Can Xenium In Situ Gene Expression data be generated from PBMCs?
Other cell types have not been tested.
In general, modifications to documentation (Demonstrated Protocols and User Guide) provided here are unsupported. We cannot guarantee assay performance even when implementing the guidance listed in this article and therefore, customers should proceed at their own risk. 10x Genomics Support will not be able to provide additional information beyond this article, details from internal experiments, or replacement reagents in the event of assay failure (cell detachment, low number of transcripts per cell).
Depending on the organism and cell type, it may be possible to culture cells on Xenium slides to obtain Xenium In Situ Gene Expression data. There are multiple considerations for this approach including slide treatment to sterilize slide and maximize cell adhesion, followed by gentle cell fixation prior to the standard Xenium In Situ Gene Expression assay workflow. The challenges of using cell samples on-slide is the potential for microbe contamination and the potential for the loss of cell adhesion. This said, we have performed a successful proof-of-concept experiment in-house using modified demonstrated protocols to show that this approach is feasible. This data is intended as a starting point for further optimization of additional cell types.
From limited testing, we have found that HEK293T cells yielded good data on the Xenium platform. Here we show an overview in Xenium Explorer of HEK293T cells processed with our human breast panel (Figure 1).
Figure 1. Xenium Explorer view of DAPI images of HEK293T after Xenium In Situ Gene Expression.
In this experiment, over 6K cells were recovered for each of two replicates and over 3M transcripts were detected with subcellular resolution and high quality scores. When comparing these data to Chromium 3’ Gene Expression data using the same cell culture stock, assay sensitivity was found to be comparable to data from 3’ gene expression library at high sequencing depth.
Methods Overview
First, Xenium slides are treated with poly-L-lysine to promote cell adhesion. Then, Xenium slides are assembled in cassettes and cell culture is plated directly onto Xenium slides. The cells are adhered to slides for ~48 h. After proliferation, cells are fixed and permeabilized (on slides) according to modified Xenium In Situ for Fresh Frozen Tissues – Fixation & Permeabilization Demonstrated Protocol (CG000581). Next, we perform the Xenium In Situ Gene Expression assay workflow (CG000582). Last, slides are loaded on the Xenium Analyzer (CG000584) for fully automated decoding and analysis.
Figure 2. High-level overview of the steps required to obtain Xenium gene expression data from cell culture.
Xenium Slide Treatment
Xenium slides and cassettes are sterilized to prevent microbe growth during the workflow and then treated with poly-L-lysine to promote cell adhesion.
Reagents and equipment:
- Poly-L-lysine solution (PLL) (Sigma Aldrich P4707-50M)
- 2 Xenium Slides (PN-3000941), part of the Slide and Sample Prep Reagent Kit (PN-1000460)
- Customers can also purchase the Xenium Slide Kit (PN-1000465) separately
- 2 Xenium Cassettes and 2 Xenium Lids, part of the Xenium Decoding Consumables (PN-1000487)
- Customers can also purchase Xenium Cassettes (PN-1000566) separately
- 1x PBS (nuclease-free, any vendor)
- Complete Media (any vendor)
- Corning® 500 mL Vacuum Filter/Storage Bottle System, 0.2 µm Pore 33.2cm² Nylon Membrane, Sterile (PN 430773)(any vendor)
- Culture dishes (any vendor)
- Forceps
- Biosafety cabinet (BSC)
- 37°C, 5% CO2 incubator
Substrates other than Poly-L-lysine solution have not been tested. Some considerations for alternative substrates are cell toxicity, thickness of coating, and potential for autofluorescence.
Preparing the slides:
Sterilize slides, cassettes, and lids with 70% ethanol followed by UV lamp treatment.
70% Ethanol Sterilization (all steps to be done inside biosafety cabinet (BSC) to maintain sterility):
- Pass Ethanol through the bottle filter.
- Use plastic culture dishes to soak the slides, cassettes, and lids in 70% ethanol.
- Soak the contents (fully submerged) for 10-15 minutes.
- Remove the ethanol from the dish and stand slides and holders against the inside of the dish to dry for 20 minutes or until fully dry.
- Lay contents into the dish and cover with dish lid.
- Close BSC and turn off the fan.
UV Sterilization:
- Turn on the UV lamp in the BSC and expose materials for 20 to 30 minutes.
- Assemble Xenium slide in the Xenium cassette according to CG000581.
- Begin the slide coating step.
- It is possible to cover the Xenium cassette with a Xenium cassette lid and place the assembly in either a 4°C fridge or 37°C, 5% CO2 incubator overnight until ready to move onto the coating step.
Coating the slides:
Coat the slide with sterilized Poly-L-lysine to promote cell adhesion:
- Remove lid from slide assembly. Add 1mL of the Poly-L-lysine coating reagent to the Xenium slide well.
- Place lid back on cassette. Place slide cassette assembly in the 37°C, 5% CO2 incubator and let sit for the respective coating time.
- Overnight recommended, but a minimum of 3 hrs time is required.
- Place slide back into the BSC.
- Remove the coating reagent from the slide using a vacuum or pipette.
- Rinse the slide with sterile PBS (repeat 3x).
- Rinse the slide with Complete Media (repeat 1x).
- Remove any excess liquid from the slide (no dry time necessary after this step.)
- Proceed immediately to plating cells.
Plating the cells:
To plate the cells, harvest and count the cells:
- Collect cells in culture and record cell count.
- Plate 0.5e5 cells/mL (1mL per slide).
- Cover with sterilized Xenium cassette lid and return to 37°C, 5% CO2 incubator.
- Adhere cells on slide for no more than 48 hours.
- If cells need to sit in the incubator for longer than 48 h, reduce the cell input to avoid overgrowth.
- Extended incubation beyond 48 h can result in overgrowth and clumps forming on the Xenium slide.
- Image slides (if necessary).
- Proceed to Xenium sample preparation.
Xenium Sample Preparation
Cultured cells on Xenium slides are ready for fixation using Xenium In Situ for Fresh Frozen Tissues – Fixation & Permeabilization Demonstrated Protocol (CG000581). Protocol modifications are required to prevent cell loss and over permeabilization.
Reagents and equipment:
- All 3rd-party reagents and equipment listed in CG000581 are required for sample preparation except for the following:
- Slide mailer (as reagents are dispensed directly in to the Xenium cassette well)
- Forceps
- SDS
- Slide drying rack
- Dry ice
- For this proof-of-concept experiment, paraformaldehyde (PFA) was used for fixation
Demonstrated Protocol:
The fixation and permeabilization protocol consists of the following steps:
All steps are performed on the prepared cell culture slides, according to the Demonstrated Protocol (CG000581), with the following exceptions:
| Step | Description | Modifications |
| 1.1 | Preparation - Buffers |
Step 1.1c
Step 1.1d
Step 1.1f
|
| 1.2 | Slide Preparation |
1.2a-d
|
| 1.3 | Fixation |
Step 1.3a
|
| 1.4 | Permeabilization |
Step 1.4a-d
Step 1.4e
Step 1.4f-h
|
Proceed immediately to Xenium In Situ Gene Expression - Probe Hybridization, Ligation & Amplification User Guide (CG000582).
Probe Hybridization, Ligation & Amplification
The Xenium Assay Workflow consists of the following steps:
All steps are performed on the prepared cell culture slides, according to the User Guide - Probe Hybridization, Post Hybridization Wash, Ligation, Amplification, Post Amplification Wash, and Nuclei Staining (CG000582). There are the following exceptions for autofluorescence quenching:
| Step | Description | Modifications |
| 5.1 | Autofluorescence Quenching |
Step 5.1a
Steps 5.1b-i
Step 5.1j-m
Step 5.1n-u
Step 5.1v-x
‘TIP’ Slides can be safely stored dry at this step (with cassette lid for up to 4 days at 4°C. Step 5.1z
|
| 5.2 | Nuclei Staining |
Step 5.2
‘!’ We have not explicitly tested a stopping point after step 5.2 and would recommend to proceed to instrument run for optimal data quality. Theoretically slides can be stored similar to slides with tissue sections: How can slides be stored prior to Xenium Analyzer run? |
Considerations for Future Optimization (for different cell types):
Here is a list of steps that may require future optimization:
-
Slide coating for optimal cell adhesion, depending on the cell type
-
Fibronectin, gelatin, collagen, etc.
-
-
Starting cell concentration
-
Permeabilization time
We cannot provide additional details on how these steps may need to be modified.
Proof of Concept Experiments - HEK293T Cell Culture
Figure 3. Experimental design used for proof of concept experiment HEK293T.
Below are observations made from a representative Analysis Summary File when running HEK293T Cell Culture through the modified Xenium assay workflow listed above and then comparing these data to the same cell culture run on the Chromium 3’ Single Cell Gene Expression assay with high sequencing depth (Figure 3).
- The Xenium data generated in this proof of concept experiment was considered to be good quality as over 6,000 cells were recovered for each of two replicates and the total number of transcripts detected ranged from ~3-5 million (Table 1).
Table 1. Key Analysis Summary metrics generated from two replicates of HEK293T cell culture. 1) Number of total cells detected for each replicate. 2) Median transcripts per cell. 3) The total number of transcripts detected. Differences between the two Xenium replicates could be attributed to the fact that this cell line has a very high RNA content, which can affect decoding efficiency of in situ methods in general. Better tailoring of panel and cell line may improve performance.
High quality decoding was observed in general, with high quality scores and a low number of probe and decoding controls (Figure 4).
Figure 4. Gene-Specific Transcript Quality. Total transcripts per gene (x) plotted by mean transcript quality of good transcripts (y). (A) Xenium Replicate 1 and (B) Xenium Replicate 2. Some genes have low quality (less than Q20), likely because they are not expressed in the cell line (low transcripts per gene).
Nuclei stained with DAPI are clearly visible and correspond well with nuclear segmentation and expansion (Figure 5). The majority of transcripts map to cells at subcellular resolution.
Figure 5. Images showing cell visualization in Xenium Explorer. (A) Nuclei with DAPI. (B) Cell segmentation through nuclear expansion. (C) Transcript localization.
Xenium data was compared to a Chromium 3’ Single Cell dataset made with the same cell culture. A high level overview of the number of cells surveyed and the UMI counts per cell for the single cell dataset are provided in Table 2. For comparisons to Xenium, a high read depth (~600k reads/cell) was selected to achieve similar sensitivity (Table 2).
Table 2. High-level overview of 3’ Single Cell feature counts. Here we show a comparable number f cells surveyed at different read depths (~20K reads per cell vs. ~600K reads per cell). The number of UMIs detected increased significantly at higher sequencing depths.
We then compared the Chromium 3’ Single Cell data to the Xenium dataset and found comparable sensitivity (Figure 5). Good correlation was observed between codeword count (Xenium data) and feature count (3’ data) (Figure 5).
Figure 5. Xenium In Situ Gene Expression and 3’ Gene Expression Count Correlation. We observe good correlation across a dynamic range. Most genes lying on the vertical point are negative controls or lowly detected genes on Xenium that are undetected in the 3’ Gene Expression assay.
Conclusions
Here we demonstrate that culturing cells on Xenium slides is feasible based on proof of concept experiments with HEK293T cells. It is possible to detect a large number of transcripts (in the range of millions) with good transcript quality. Protocol modifications are required for optimal assay performance.
Product: Xenium In Situ Gene Expression, Xenium Prime In Situ Gene Expression