Method for separating clusters of malignant cells and clusters from stromal cells of a malignant tumour tissue sample

摘要

Ex vivo separation of malignant cells clusters and stromal cells clusters from a malignoma tissue sample, comprises (a) cleaning and crushing the malignoma tissue sample into pieces of 1-4 mm 3, (b) suspending the minced tissue in a culture medium, (c) subjecting the resulting suspension to a treatment with collagenase (i.e. a collagenase digest) at 36-37[deg] C, (d) centrifuging obtained collagenase digestion product for 5 minutes at 50x g at room temperature, (e) separating pellet and the supernatant from the product obtained in (d). Separating malignant cells clusters and stromal cells clusters from a malignoma tissue sample in ex vivo, comprises (a) cleaning and crushing the malignoma tissue sample into pieces of 1-4 mm 3, (b) suspending the minced tissue in a culture medium, (c) subjecting the resulting suspension in (b) to a treatment with collagenase (i.e. collagenase digest) at 36-37[deg] C, (d) centrifuging obtained collagenase digestion product in (c) for 5 minutes at 50x g at room temperature, (e) separating pellet and the supernatant from the product obtained in (d), (f) pipetting and re-suspending the pellet from (e) in the culture medium by an absorbing instrument, preferably by a pipette tip for several times, preferably 4-6 times, and pipetting and transferring into a culture vessel, (g) analyzing the re-suspension obtained in (f) by using a microscope (preferably an inverted microscope with phase optics) with respect to at least one cell association, in which an association of malignant cells can be identified by its phenotypic appearance, or an association of endothelial cells can be identified by its phenotypic appearance, or an association of fibroblasts can be identified by its phenotypic appearance and (h) separating the identified cell clusters. Independent claims are also included for (1) checking the response of malignant cells of the malignoma tissue sample in ex vivo by chemotherapeutic agent and/or radiation therapeutic agent, comprising subjecting the malignoma tissue sample into the process as mentioned above and analyzing the cells that are identified as malignant cell clusters; and (2) checking the growth inhibitory effect of chemotherapeutic agent and/or radiation therapeutic agent on the malignoma tissue sample in ex vivo, comprising incubating the chemotherapeutic agent and/or radiation therapeutic agent with the malignoma tissue sample, analyzing the number of cells and/or cell colonies, and determining the IC50 value of the chemotherapeutic and/or radiation therapeutic agent, where the malignoma tissue sample is digested into stromal cells and malignant cells using the above mentioned method, before incubating with the chemotherapeutic agent and/or radiation therapeutic agent and only malignant cells is incubated with the chemotherapeutic agent and/or radiation therapeutic agent and IC50 value of the drug is analyzed. ACTIVITY : Angiogenesis inhibitor; Cytostatic. No biological data given. MECHANISM OF ACTION : None given.