title: A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.
abstract: 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.
  
  
  
  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 °C to overnight dissociation at ambient temperature.
  
  
  
  The use of NP allows for the most effective dissociation of viable single cells from human BTs or brain tissue. Its non-aggressive dissociative capacity may enable ambient-temperature shipping of tumor pieces in multi-center clinical trials, meanwhile being dissociated. As clinical grade NP is commercially available it can be easily integrated into cell-therapy clinical trials in neuro-oncology. The high quality viable cells produced may enable investigators to conduct more consistent research by avoiding the experimental artifacts associated with the presence dead cells or cellular debris.
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel. volovitz@yahoo.com.|Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel. volovitz@yahoo.com.
authors.forename: Ilan
authors.initials: I
authors.lastname: Volovitz
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Netanel
authors.initials: N
authors.lastname: Shapira
authors.affiliation: Department of Neurosurgery, Galilee Medical Center, Lohamei HaGeta'ot 5, Nahariya, Israel.
authors.forename: Haim
authors.initials: H
authors.lastname: Ezer
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Aviv
authors.initials: A
authors.lastname: Gafni
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Merav
authors.initials: M
authors.lastname: Lustgarten
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Alter
authors.affiliation: Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Idan
authors.initials: I
authors.lastname: Ben-Horin
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Ori
authors.initials: O
authors.lastname: Barzilai
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Shahar
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Andrew
authors.initials: A
authors.lastname: Kanner
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Itzhak
authors.initials: I
authors.lastname: Fried
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Igor
authors.initials: I
authors.lastname: Veshchev
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Rachel
authors.initials: R
authors.lastname: Grossman
authors.affiliation: Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Ram
mesh_terms: D001426:Bacterial Proteins; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D047009:Hathewaya histolytica; D004798:Enzymes; D005615:Freezing; D005910:Glioma / Q000378:metabolism / Q000473:pathology; D006801:Humans
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes
keywords: Brain; Brain metastasis; Brain tumors; Collagenase; DNase; Dispase; Glioblastoma; Glioma; Neutral protease; Tissue dissociation