A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells

BMC Neuroscience

Methodology Article

Idan Ben-Horin¹  Aviv Gafni¹  Tal Alter¹  Netanel Shapira¹  Merav Lustgarten¹  Ilan Volovitz²  Haim Ezer³  Tal Shahar⁴  Rachel Grossman⁴  Itzhak Fried⁴  Zvi Ram⁴  Igor Veshchev⁴  Ori Barzilai⁴  Andrew Kanner⁴ 

[1] Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel;Cancer Immunotherapy Laboratory, Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel;Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel;Department of Neurosurgery, Galilee Medical Center, Lohamei HaGeta’ot 5, Nahariya, Israel;Department of Neurosurgery, Tel Aviv Sourasky Medical Center, Weizmann 6, Tel Aviv, Israel;

关键词: Brain tumors;    Glioma;    Glioblastoma;    Brain metastasis;    Brain;    Tissue dissociation;    Neutral protease;    Dispase;    Collagenase;    DNase;   

DOI  :  10.1186/s12868-016-0262-y

 received in 2016-01-12, accepted in 2016-05-11,  发布年份 2016

来源: Springer

【 摘 要 】

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.ResultsOver 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© The Author(s) 2016