Journal article
A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells
BMC Neuroscience, Vol.17, 30
Jun/2016
Abstract
Background: Conducting research on the molecular biology, immunology, and physiology of brain tumors (BTs) and primary brain tissues requires the use of viably dissociated single cells. Inadequate methods for tissue dissociation generate considerable loss in the quantity of single cells produced and in the produced cells' viability. Improper dissociation may also demote the quality of data attained in functional and molecular assays due to the presence of large quantities cellular debris containing immune-activatory danger associated molecular patterns, and due to the increased quantities of degraded proteins and RNA. Results: Over 40 resected BTs and non-tumorous brain tissue samples were dissociated into single cells by mechanical dissociation or by mechanical and enzymatic dissociation. The quality of dissociation was compared for all frequently used dissociation enzymes (collagenase, DNase, hyaluronidase, papain, dispase) and for neutral protease (NP) from Clostridium histolyticum. Single-cell-dissociated cell mixtures were evaluated for cellular viability and for the cell-mixture dissociation quality. Dissociation quality was graded by the quantity of subcellular debris, non-dissociated cell clumps, and DNA released from dead cells. Of all enzymes or enzyme combinations examined, NP (an enzyme previously not evaluated on brain tissues) produced dissociated cell mixtures with the highest mean cellular viability: 93 % in gliomas, 85 % in brain metastases, and 89 % in non-tumorous brain tissue. NP also produced cell mixtures with significantly less cellular debris than other enzymes tested. Dissociation using NP was non-aggressive over time-no changes in cell viability or dissociation quality were found when comparing 2-h dissociation at 37 degrees C to overnight dissociation at ambient temperature. Conclusions: The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacit
Details
- Title
- A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells
- Creators
- Ilan Volovitz (null)Netanel Shapira (null)Haim Ezer (null)Aviv Gafni (null)Merav Lustgarten (null)Tal Alter (null)Idan Ben-Horin (null)Ori Barzilai (null)Tal Shahar (null)Andrew Kanner (null)Itzhak Fried (null)Igor Veshchev (null)Rachel Grossman (null)Zvi Ram (null)
- Resource Type
- Journal article
- Publication Details
- BMC Neuroscience, Vol.17, 30; Jun/2016
- Number of pages
- 10
- Language
- English
- DOI
- https://doi.org/10.1186/s12868-016-0262-y
- Grant note
- Public Committee for Allocation of Estate Funds, Ministry of Justice, Israel [5313]_ALMAME_DELIMITER_
- Scientific Unit
- The Weizmann Institute of Science
- Record Identifier
- 993262121903596
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