Pioneering 10x Flex on OCT-Embedded Patient Brain Tissue

Frozen, fixed, and embedded tissue is omnipresent in medical research. A scientist may sometimes perform (immuno-)histological research on fresh tissue, but logistics, planning and the technological infrastructure of labs usually necessitate a preservation, fixation, and/or embedding step. Such a step enables flexibility in timeline, transport, long-term storage, and overall user-friendliness.

Moreover, embedding tissue in paraffin or optimal cutting temperature (OCT) compound fits tissues into blocks that can be safely sectioned into slices. Traditionally, slices are the microscopists’ perfect research format. Many subsequent, modern research technologies, therefore, are adapted to work on embedded slices, too. However, single-cell sequencing on OCT-embedded tissue was not performed much until the launch of 10x Flex.

Our 10x Genomics Single Cell Gene Expression Flex service (10x Flex in short) namely makes it possible to perform high-throughput single-cell RNA sequencing on frozen and formaldehyde-fixed tissue. Formaldehyde-fixed paraffin-embedded (FFPE) tissue was already known to be compatible with this technology. With a protocol recently performed for clients for the first time, we have experienced that OCT-embedded tissue is, too.

 

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This case study details the successful establishment of 10x Flex on OCT-embedded tissue for clients of the Neuropathology department of Amsterdam UMC, who work with resected brain tissue of epilepsy patients.

Optimal Cutting Temperature

Optimal Cutting Temperature (OCT) refers to a protocol that uses the OCT compound, a water-soluble and quickly frozen compound that forms a solid block encasing the tissue. This solid block can then be sectioned using a cryostat, a precise microtome slicer in a frozen environment, and mounted on slides for microscopic analysis. The OCT compound helps to prevent artifacts or damage that may occur during the freezing and cutting processes, ensuring better preservation of tissue architecture, cellular details, and RNA.

 

Frozen tissue (non-brain) in a cryostat apparatus.

 

Surgically removed tissue ends up as OCT-embedded tissue if necessary for future experimental steps. Directly during surgery, the tissue is snap-frozen in liquid nitrogen and then embedded in OCT to ensure minimal quality loss during slicing. Precious as patient material usually is, embedded tissues are usually used for research purposes as efficiently as possible. 

“We perform various lines of research on brain tissue from patients with the rare genetic disease Tuberous Sclerosis Complex.” Says Mirte Scheper, PhD candidate in the group of Prof. Eleonora Aronica at Amsterdam UMC. “Almost all patients have epilepsy along with other neuropsychiatric comorbidities and will undergo resection brain surgery.” The resected material is snap-frozen and partially OCT, partially paraffin-embedded. The paraffin-embedded material is used, for example, for immunohistochemical staining. “And we aimed to perform single-cell sequencing on the OCT-embedded material,” she adds.

Reasons for single-cell sequencing

Tuberous Sclerosis Complex (TSC) is a rare, genetic disease that causes benign neoplasms to grow in the central nervous system. As Scheper explained previously, epilepsy is one of the symptoms of the disease.

Scheper and colleagues are investigating the hypothesis that TSC patients’ GABAergic interneurons fail to switch to an inhibitory state during development. This would disrupt the excitatory/inhibitory balance in the brain. “However,” she says, “we’re not sure which specific subtype of GABAergic interneuron this involves, and there are quite a few different varieties of subtypes.”

 

Epilepsy in Tuberous Sclerosis Complex may be driven by genetic disfunction based around the mTORC1 protein. One of the studied traits of the disease is a network imbalance (top right) dysregulation of mTOR signaling itself results in developmental alterations of the balance between excitation and inhibition. Source: Aronica, E. et al. (2023)

 

“Therapeutically, we see that all the medication is very general,” she explains. “Most antiseizure medications either increase inhibition or decrease excitation in the whole brain. The medication, taken orally, affects all cells but does not involve every cell type. Parts of the brain unaffected by the disease are still affected by the therapy, which means that normal communication and functioning of the brain are affected in areas where they shouldn’t be. I think that if we can identify the immature interneuron subtypes in TSC, we can start targeting cell types and increase specificity.”

If we can identify the immature interneuron subtypes in Tuberous Sclerosis Complex, we can target cell types and increase drug specificity.

Scheper: “In the disease I work on, up to 60% of the children don’t respond to anti-seizure medication properly. That indicates that there’s a need for a new therapy—and also a new angle of research—that prioritizes high target specificity. Since you can get the gene expression information on specific subtypes with single-cell sequencing, that was the most logical technology for this purpose.”

Reasons for 10x Flex

As mentioned, there was not yet an established protocol for single-cell sequencing on the specific material. “Because the material is so rare, we wanted it to incur the least damage as possible for analysis. For that reason, we did not want to cut thicker slices and do single-cell sequencing on those slices, as that could injure too much material.” Hence, they looked for a way to perform single-cell sequencing on the OCT-embedded sections that were natural to the lab’s infrastructure.

10x Genomics Single Cell Flex (10x Flex) is a kit by 10x Genomics that enables single-cell RNA profiling on frozen, formaldehyde-fixed, and embedded tissue. Fresh and cryopreserved cells can be formaldehyde fixed with a fixation protocol so that it is compatible with the single-cell sequencing library preparation that follows.

 

After sample preparation, 10x Flex libraries are run on the Chromium X

 

“10x Genomics actually now recommends 10x Flex as the new standard for tissue like ours,” Scheper explains. “We had done single nucleus sequencing previously, which works a bit differently, so we were interested whether with 10x Flex, sequencing would be possible indeed.”

Recommendations from a co-worker in Utrecht led her to Single Cell Discoveries. After an exploratory call with our PhD-level scientists, the 10x Flex recommended protocol for OCT tissue was indeed determined to be the best fit. “All the grants that our group is writing do involve some form of single-cell sequencing, so we were eager to try a new technology that might be the best fit for the future,” Scheper says.

Piloting and success

To avoid putting too much precious material on the line, the team started with two pilot analyses, where they submitted sections from two OCT-embedded blocks. Nataša Josipović, PhD, led the bespoke service experiments at Single Cell Discoveries.

“Already for the first pilot, the prepared samples went through all our quality controls,” she explains. This was repeated for the second pilot, which opened the door for a larger input. Sixteen samples, from tissue covering two different diseases, followed, and later, five more from co-workers at Utrecht UMC with yet a different disease. All went through quality control and resulted in quality single-cell data.

“We were definitely a bit surprised,” Josipović says. “This was our first time seeing this type of sample. But we ended up getting plenty of workable material from the OCT sections for sequencing. Best of all, the remaining cells are now safely fixed and stored and can be used again for another 10x Flex analysis or even immunofluorescence.”

All our current grants involve some form of single-cell sequencing, so we were eager to try a new technology that might be the best fit for the future.

Scheper will be analyzing the data in the future to study the GABAergic interneuron subtypes of her interest. She looks back on a fruitful collaboration. “It was really nice. The amount of specialized knowledge at Single Cell has been very helpful. It’s a very non-stressful way of retaining so much information to work with people who have actual knowledge of what they are doing. It brought us a very easy and professional way to sequence our material and gain more information on our disease.”