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Isolation of viable primary cells and intact nuclei for downstream analysis

We offer state of the art techniques to enrich the postmortem brain tissue, including isolation of primary cells and intact nuclei. This uniquely enables bulk, single-cell or -nucleus transcriptomics as well as protein profiling.

CNS-resident immune cell isolation 

Our core expertise is in the isolation of primary CNS-resident immune cells including microglia, T cells and B cells. Pure CNS-resident immune cells are isolated using a rapid procedure based on Percoll density gradient centrifugation followed by magnetic beads-positive selection or fluorescence activated cell sorting.  Due to a short postmortem delay and rapid procedure the primary cells show preservation of gene expression and phenotype that reflects neurological diagnosis.

Our isolated primary cells can be provided fresh in culture medium for use in cellular assays, as well as frozen, or lysed, enabling downstream molecular characterization as demonstrated in multiple publications making use of NBB tissue (see Publication list below).

Microglia isolation using percoll density gradient centrifugation and magnetic beads-positive selection

Isolation and sorting of nuclei

Nuclei are isolated from frozen tissue from the NBB collection by staining with cell type specific markers such as IRF8+ (microglia) or NeuN (neurons) followed by fluorescence-activated nuclei sorting. This enables to isolate cell-type specific RNA sequencing analyses and single-nucleus sequencing. By linking these molecular data to the neuropathological data from the NBB donors we support identification of alterations in RNA expression that link to a disease state. As such we strive to contribute to identification and validation of disease pathways and novel targets.

Procedure for fluorescence-activated nuclei sorting

 

Tissue characterization

Tissue characterization procedures may include histological or immunohistochemical analysis to enrich the pathological donor data. We apply optimized protocols to visualize common hallmarks of disease such as βA4, α-syn, TDP43 and offer to validate and apply customized protocols for staining of specific targets of interest in fixed or freshly frozen tissue. In addition, other quantitative methods for expression of RNA or proteins are offered to explore the expression of potential therapeutic targets. Together this will facilitate optimal selection and characterization of tissue, at the level of the donor or tissue block.

Example histological and immunohistochemical staining for neuropathological assessment of donors

 

Custom dissection and prospective collection of tissue

The NBB obtains for all donors approximately 90 different samples including tissue from different brain regions, cerebrospinal fluid (CSF), buffy coat and plasma. From all donors we store frozen and FFPE tissue blocks. In addition to the standard dissection protocols we offer custom dissection procedures including manual dissection and laser capture dissection. Prospective tissue collection procedures enable to deliver fresh tissue and allow for alternative fixation methods or tissue block dimensions.

 

Consultancy Services – Donor selection and methodological advice

Quality research on post-mortem tissue requires careful study design, selection of donors, tissue and experimental approach. To facilitate the full process from initial target discovery and validation to preclinical research we give tailored advice throughout all these steps along the therapeutics development path. In particular we share our expertise in post-mortem tissue handling and study design, selection of donors and tissues and neuropathological examination of tissue morphology and histology.

 

Publications

Our products and services are built on validated protocols and scientific expertise of the NBB staff. Overview of reference literature:

1. Böttcher C, van der Poel M, Fernández-Zapata C, Schlickeiser S, Leman JKH, Hsiao CC, et al. Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis. Acta Neuropathol Commun. 2020 Aug 18;8(1):136.

2. Mizee MR, Poel M van der, Huitinga I. Purification of cells from fresh human brain tissue: primary human glial cells. In: Handbook of Clinical Neurology [Internet]. Elsevier; 2018 [cited 2022 Jan 24]. p. 273–83. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780444636393000190

3. van der Poel M, Ulas T, Mizee MR, Hsiao CC, Miedema SSM, Adelia  null, et al. Transcriptional profiling of human microglia reveals grey-white matter heterogeneity and multiple sclerosis-associated changes. Nat Commun. 2019 Mar 13;10(1):1139.

4. Melief J, Koning N, Schuurman KG, Van De Garde MDB, Smolders J, Hoek RM, et al. Phenotyping primary human microglia: tight regulation of LPS responsiveness. Glia. 2012 Oct;60(10):1506–17.

5. Lopes K de P, Snijders GJL, Humphrey J, Allan A, Sneeboer MAM, Navarro E, et al. Genetic analysis of the human microglial transcriptome across brain regions, aging and disease pathologies. Nat Genet. 2022 Jan;54(1):4–17.

6. Fransen NL, Hsiao CC, van der Poel M, Engelenburg HJ, Verdaasdonk K, Vincenten MCJ, et al. Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions. Brain J Neurol. 2020 Jun 1;143(6):1714–30.

7. Hendrickx DAE, van Scheppingen J, van der Poel M, Bossers K, Schuurman KG, van Eden CG, et al. Gene Expression Profiling of Multiple Sclerosis Pathology Identifies Early Patterns of Demyelination Surrounding Chronic Active Lesions. Front Immunol [Internet]. 2017 [cited 2022 Apr 25];8. Available from: https://www.frontiersin.org/article/10.3389/fimmu.2017.01810

8. Michailidou I, Willems JGP, Kooi EJ, van Eden C, Gold SM, Geurts JJG, et al. Complement C1q-C3-associated synaptic changes in multiple sclerosis hippocampus. Ann Neurol. 2015 Jun;77(6):1007–26.

9. Geut H, Hepp DH, Foncke E, Berendse HW, Rozemuller JM, Huitinga I, et al. Neuropathological correlates of parkinsonian disorders in a large Dutch autopsy series. Acta Neuropathol Commun. 2020 Mar 26;8(1):39.

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