Almost all neural cell cultures must have macromolecules as growth substrates to which the cells adhere in order to grow. Commonly used are polylysine and polyornithine. There are also some extracellular matrix (ECM) components such as laminin, fibronectin, and collagen. Source: Laboratory Techniques in the Use of Neurobiology
Operation method
basic program
Principle
Almost all neuronal cell cultures must have macromolecules as growth substrates to which the cells adhere in order to grow. The most commonly used are polylysine and polyornithine. In addition, there are some extracellular matrix (ECM) components, such as laminin, fibronectin, and collagen.
Materials and Instruments
Neural Cells Move 1.Polylysine and polyornithine; Polylysine and polyornithine are two of the most commonly used macromolecules to promote cell-adherent growth.Polylysine or polyornithine can be ordered from various reagent companies.Poly-L-lysine hydrobromide (SigmaP6282) is commonly used in our lab.It is prepared in boric acid buffer pH 8.4 or PBS pH 7.4 to 100µg/ml,filtered and dispensed. (SigmaP6282), formulated into 100µg/ml with pH 8.4 boric acid buffer or pH 7.4 PBS, filtered and dispensed, and stored at -20℃. When encapsulating the culture plate, we usually use 25µg/ml concentration for encapsulation, 37℃ for 4h or overnight, and then wash with sterile water for 3 times (make sure to wash away the unbound macromolecules because they are cytotoxic), and then air-dry in the ultra-clean bench. The encapsulated culture plates and cell vials can be stored at 4℃ for 2-4 weeks, and the polylysine can be recycled and reused 1-2 times. 2.Extracellular matrix components (ECMcomponents). ---------- ---------- ---------- -------- Extracellular matrix components are the most important involved in intercellular adhesion and contact in vivo The binding of extracellular matrix components to cell surface receptors not only regulates cell adhesion, but also directly regulates cell metabolism, differentiation and gene expression. It is suitable for neuronal culture, especially for peripheral nervous system neurons. Collagen is the most abundant protein in the animal body, type I fibrillar collagen has been used in the tissue culture of the nervous system for a long time, and it is still commonly used in the culture of neurons of the peripheral nervous system and cell lines of the nervous system. Type I fibrillar collagen can be extracted from rat tails and is now available commercially. Collagen can be coated by covering the culture medium with a very small amount of the solution and leaving it on an ultra-clean bench to evaporate and solidify naturally. After collagen, fibronectin and laminin are the other two components of the extracellular matrix used for neuronal cell culture in vitro. Different cell types have different sensitivities to the two proteins, and neurons tend to grow better on laminin-coated matrices. Laminin (Sigma L2020) and fibronectin (Sigma F0635) were encapsulated at concentrations of 10µg/ml and 20µ,g/ml, respectively. 3. 3. Adhesive growth of neural stem cells Neural stem cells are usually grown in spherical suspension, but for adherent culture, 0.001 % poly(ornithine) and 20 µ,g/ml of fibronectin (Sigma F0635) can be used. For adherent culture, 0.001 % ornithine and 0.2% gelatin can be used to coat new culture flasks overnight with ornithine and 4-5h with gelatin, then the gelatin can be sucked out and the neurospheres can be added, and on the second day, it can be observed that the neural stem cells grown in spheres have already been grown in a monolayer. Caveat 1. The water quality of the configured reagents should be guaranteed, and it is better to use ultra-pure water, and the preparation should be precise. If you have the conditions, you can choose the commercialized reagents that have been configured. 2. All utensils and instruments should be clean, dry and sterile. Do not use detergent to clean the utensils used, and all glassware should be over acid. Our laboratory now uses a new type of detergent "Dikang 90", which can be decomposed biologically decreasing, completely washable and not easy to burn, instead of mixing the detergent with acid, to ensure the safety of the staff. 3. When adding antibiotics to serum-free medium, the concentration used in serum medium should be reduced by at least 50%. Because serum proteins bind and inactivate some antibiotics, which are not inactivated under serum-free culture conditions, too high a concentration may act as a toxic level for the cells.4 With the use of any antimitotic agent, its neuronal toxicity must be considered, and the lowest effect concentration for use should be determined. For example, cytarabine can be toxic to certain types of neurons at very low concentrations and can cause death of specific neurons. Other anti-mitotic agents have not demonstrated such toxicity. 5. Additives and reagents should not be repeatedly freeze-thawed, especially those containing proteins. Excessive freezing and thawing will affect their biological activity. 6. Care should be taken when preparing digestive enzyme solutions to use an appropriate solution (free of Ca2+, MgMg2+, Mg), adjusted to the appropriate pH. Dissociation of cells by enzyme digestion is generally less damaging to the cells than mechanical dispersion, especially for the more highly differentiated cells with irregular morphology (e.g., adult animal neural tissue cells). However, over-digestion of cells with trypsin solution can also cause greater damage to cells. Therefore, care must be taken to control the enzyme plant (enzyme concentration), the duration of action, and the temperature of the enzyme treatment. Common Problems Experimental tips for culturing nerve cells: 1.Selection of medium is a basic and important task in cell culture. This can be done by consulting the references, or by asking for advice when purchasing cell lines. It may be worthwhile to try the media commonly used in this lab, many of which can be suitable for a wide range of cells. 2 Exactly which digestive enzyme solution to use depends on the specific composition of the extracellular mesenchyme of the tissue to be dissociated. In addition, it is often necessary to combine several digestive enzymes depending on the tissue. For example, trypsin solution is used alternately with collagenase solution, and collagenase solution is used in combination with hyaluronidase. More often than not, enzymatic digestion is used in combination with mechanical separation to dissociate the cells. 3. When cleaning the crawler tablets by yourself, it is better to put the crawler tablets in a large flat dish and put it on a horizontal shaker to clean them. 4. In the primary culture of many neurons, the method of co-coating can be used. After polylysine or polyornithine coating, laminin or other extracellular matrix components can be used to achieve the best effect, and the macromolecules can promote the combination of extracellular matrix components with the culture plate. 5. The concentration and duration of action of the various coating reagents are subject to many different interpretations. Different laboratories have different systems, and it is still necessary for the experimenters to figure out by themselves. Although the coated plates can be stored at 4°C for a period of time, it is better to use the coated plates now when culturing some special neurons. For more product details, please visit Aladdin Scientific website.
Culture fluid
Culture Bottle Thermostat