Ex-vivo models
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To study the functional role of CDR proteins as well as the pathological role of CDR antibody binding we established three ex-vivo models:
- Cancer cell lines
- Rat Purkinje neuron culture (PNC)
- Rat Cerebellar organotypic slice culture (cOTSC)
Cancer cell lines:
HepG2, OvCar3, OvCar4 and BT474 expressing either both CDR2 and CDR2L or only CDR2 at the endogenous levels. This experimental model system is less sensitive then the neuronal primary culture of Purkinje neurons and it is easy to manipulate by utilizing techniques such as CRISPR-Cas9 to knockout the CDR proteins. We are using these cell lines to study protein location and protein-protein interaction.
Purkinje neuron culture (PNC):
PNC is a heterogeneous primary culture of cerebellar cells with enrichment on Purkinje neurons, the main target in PCD. To obtain a high population of Purkinje neurons can be difficult and exhausting process, because they are extremely sensitive and need additional supplements/nutrients for survival. We mainly use this “single neuronal cell level” model to place/evaluate findings regarding CDR protein location and protein-protein interactions obtained in the cancer cell line studies in neuronal prospective. But also to study anti-Yo and compounds effects on Purkinje neuron morphology.
Cerebellar organotypic slice culture (cOTSC):
OTSC can be used to study neurochemical, structural and physiological changes linked to diseased in vivo brains. Hence, we created two ex-vivo antibody-mediated PCD models by applying anti-Yo (CDR2/2L) positive patient sera (immuno-response and antibody-mediated pathology) and affinity-purified rabbit CDR antibodies (antibody-mediated pathology) to the culture medium of rat cOTSC. These slices have the unique advantages that the tissue architecture is preserved, synaptic circuitries are maintained, and various experimental approaches including compound studies can be evaluated with or without the influence of activated immune cells and the blood-brain barrier regarding PCD pathology.