A new protocol for cultivation of predegenerated adult rat Schwann cells

Impairment of peripheral neurons by anti-cancer agents, including taxanes and platinum derivatives, has been considered to be a major cause of chemotherapy-induced peripheral neuropathy (CIPN), however, the precise underlying mechanisms are not fully understood. Here, we examined the direct effects of anti-cancer agents on Schwann cells. Exposure of primary cultured rat Schwann cells to paclitaxel (0.01 μM), cisplatin (1 μM), or oxaliplatin (3 μM) for 48 h induced cytotoxicity and reduced myelin basic protein expression at concentrations lower than those required to induce neurotoxicity in cultured rat dorsal root ganglion (DRG) neurons. Similarly, these anti-cancer drugs disrupted myelin formation in Schwann cell/DRG neuron co-cultures without affecting nerve axons. Cisplatin and oxaliplatin, but not paclitaxel, caused mitochondrial dysfunction in cultured Schwann cells. By contrast, paclitaxel led to dedifferentiation of Schwann cells into an immature state, characterized by increased expression of p75 and galectin-3. Consistent with in vitro findings, repeated injection of paclitaxel increased expression of p75 and galectin-3 in Schwann cells within the mouse sciatic nerve. These results suggest that taxanes and platinum derivatives impair Schwan cells by inducing dedifferentiation and mitochondrial dysfunction, respectively, which may be important in the development of CIPN in conjunction with their direct impairment in peripheral neurons.

Section: Discussionmentioning

confidence: 62%

Taxanes and platinum derivatives impair Schwann cells via distinct mechanisms

Imai

Koyanagi

Azimi

et al. 2017

“…SC represent mature, differentiated cells, which is why almost no risk of uncontrolled proliferation exists. An additional advantage is that isolating and culturing them is a simple process 65 . Predegenerated Schwann cells might be isolated from each of the peripheral nerves, so they might be prepared and applied also as autotransplants; the best quality SC tend to originate from mixed (i.e., sensory-motor) nerves 53 54 .…”

Section: Discussionmentioning

confidence: 99%

“…After collection of nerves, animals were sacrificed. The nerves were cultured on 12-well plates at 37 °C in humidified air with 5% CO 2 in Endothelial Cell Growth Medium (EGM2MV BulleKit, Lonza, Basel, Switzerland) supplemented with 10 μg/ml insulin (Sigma, St. Louis, MO, USA), 12 ng/ml heregulin α (Sigma, St. Louis, MO, USA), 3 µM forskolin (Sigma, St. Louis, MO, USA) and 10 μg/ml pituitary bovine extract (Sigma, St. Louis, MO, USA) as described before 65 . The cells, after the third split, were double-stained with anti-S100 antibody (dilution 1:300) and anti-GFAP antibody (dilution 1:1000).…”

Section: Methodsmentioning

confidence: 99%

Predegenerated Schwann cells–a novel prospect for cell therapy for glaucoma: neuroprotection, neuroregeneration and neuroplasticity

Smędowski

Liu

Pietrucha‐Dutczak

et al. 2016

Self Cite

Glaucoma is an optic neuropathy that leads to irreversible blindness. Because the current therapies are not sufficient to protect against glaucoma-induced visual impairment, new treatment approaches are necessary to prevent disease progression. Cell transplantation techniques are currently considered to be among the most promising opportunities for nervous system damage treatment. The beneficial effects of undifferentiated cells have been investigated in experimental models of glaucoma, however experiments were accompanied by various barriers, which would make putative treatment difficult or even impossible to apply in a clinical setting. The novel therapy proposed in our study creates conditions to eliminate some of the identified barriers described for precursor cells transplantation and allows us to observe direct neuroprotective and pro-regenerative effects in ongoing optic neuropathy without additional modifications to the transplanted cells. We demonstrated that the proposed novel Schwann cell therapy might be promising, effective and easy to apply, and is safer than the alternative cell therapies for the treatment of glaucoma.

“…In most published protocols fibroblast elimination has been achieved through various methods that exploit the differential biological properties of SCs and fibroblasts. These methods have used selective growth media101340414243, anti-mitotic drugs231, differential adhesion to a plastic substrate3111244, and immunoselection26273145. Here, we have optimized two independent protocols for SC purification by either positive or negative MACS based on cell surface labeling with p75 NGFR or Thy-1 antibodies, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…This step, which is intended to allow Wallerian degeneration to take place while concomitantly allowing SC dedifferentiation, proliferation and myelin degradation, has been shown to increase both the viability and yields of SCs obtained from adult nerves691011121314. It has also been argued that in vitro pre-degeneration of adherent nerve tissue explants promotes the outgrowth of fibroblasts and contributes to reduce fibroblast contamination in the initial populations11.…”

mentioning

confidence: 99%

A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves

Andersen

Srinivas

Piñero

et al. 2016

We herein developed a protocol for the rapid procurement of adult nerve-derived Schwann cells (SCs) that was optimized to implement an immediate enzymatic dissociation of fresh nerve tissue while maintaining high cell viability, improving yields and minimizing fibroblast and myelin contamination. This protocol introduces: (1) an efficient method for enzymatic cell release immediately after removal of the epineurium and extensive teasing of the nerve fibers; (2) an adaptable drop-plating method for selective cell attachment, removal of myelin debris, and expansion of the initial SC population in chemically defined medium; (3) a magnetic-activated cell sorting purification protocol for rapid and effective fibroblast elimination; and (4) an optional step of cryopreservation for the storage of the excess of cells. Highly proliferative SC cultures devoid of myelin and fibroblast growth were obtained within three days of nerve processing. Characterization of the initial, expanded, and cryopreserved cell products confirmed maintenance of SC identity, viability and growth rates throughout the process. Most importantly, SCs retained their sensitivity to mitogens and potential for differentiation even after cryopreservation. To conclude, this easy-to-implement and clinically relevant protocol allows for the preparation of expandable homogeneous SC cultures while minimizing time, manipulation of the cells, and exposure to culture variables.