Neurons (also called neurons or nerve cells) are the fundamental units of the brain and nervous system, the isolation and culture of nervous in vitro has proven to be very powerful approach in addressing the neurobiological process in neuroscience research. Primary neuron cultures are powerful model systems used to study neuron morphology and differentiation, synaptic function and neurotransmitter release, neurotoxicity during pre-clinical drug development, and disease modeling. As neurons do not divide and proliferate again after separation, establish an easy, reproducible, and cost-effective method to isolate and culture the primary nerve cells would greatly advance neuroscience research.

However, primary neurons are highly sensitive to isolation conditions and their culture or growth environment. Because methods differ from lab to lab, cell yields, viability, and maturation rates are highly variable and make it difficult to compare results and repeat experiments between laboratories.

 So, how to quickly prepare high quality rat/mouse nerve cell suspension samples? 

Compared with newborn mice, the dissociation of adult rat brain is more complex, requiring a combination of mechanical and enzymatic hydrolysis to degrade extracellular matrix. Typically, removal of the myelin requires a prolonged incubation period with digestion enzymes, severe mechanical disintegration, and/or additional steps for myelin purification, which altogether compromises the recovery and viability of primary cell suspensions.

In this protocol, we describe a method for isolating mouse neural cells using gentle tissue digestion enzyme formulation. The detailed experimental instruments, materials, and experimental procedure are listed as follow.

Experimental instruments and materials

RWD DSC-400 Single Cell Suspension Dissociator;
RWD HJ-400 Heating jacket;
RWD DHABE-5003 High Activity Adult Brain Enzymatic Digestion Kit(Mouse and Rat);
RWD SCT-100 Tissue processing tube;
70μm Cell filter;
RWD Animal surgical instrument;
Cell culture supplies; Pipette.

Experimental procedure 

1. According to the instructions, mix the enzyme MIX and activate it with water bath at 37℃.
2. The brain tissues of adult rats (P>7) were dissected and temporarily stored in petri dishes containing HBSS (Ca2+ and Mg2+). The blood vessels of the brain tissues were gently removed as much as possible with -tweezers

3. Put the brain tissue and enzyme MIX into the tissue processing tube, and cut the whole brain into 4 pieces with scissors.
4. Invert the tissue treatment tube to the single cell suspension dissociator, install the heating jacket, and run the program M_ABrain_Heater_2.
5. Cell suspension samples were filtered with a cell filter and centrifuged for 10 min at 300×g at room temperature.

6. Transfer the cell suspension to a 15mL centrifuge tube, add the defragmentation reagent, mix well, lay the upper layer with pre-cooled PBS and spin at 3000×g for 10 min at 4℃ with speed up 5 and speed down 3.The keep the lower layer of cell pellets, wash and collect.

7. Crack red operation (optional).
8. Cell count and flow cytometry

Experimental result

The cells of adult mouse brain tissue processed by RWD single cell suspension dissociator apparatus can reach 90% cell viability, which is ideal percentage for the downstream experiment. RWD DHABE-5003 Mouse Adult Brain Tissue Gentle Enzymolysis Kit can be used for single cell suspension dissociator of entire brain tissue of adult rat or mouse (P>7).It also can support the isolation of all neural and non-neural cells in the brain.