Positive control substance for in vitro cytotoxicity test

bodyPositive control substance for extracellular cytotoxicity testfromHatano Research institute (HRI)Research production，Widely used in vitro cytotoxicity experiments, it can meet the requirements of the Chinese Pharmacopoeia-Cell toxicity testing methodsISO 10993andYBB 00012003Parameter requirements for cytotoxicity testing method.

Positive control substance for in vitro cytotoxicity testUse conventional culture medium for extraction. The extraction method is as follows: sterile operation, take1gPositive control substance for in vitro cytotoxicity testplaced in15mLIn a sterile centrifuge tube, according to3mL/gAdd the extraction ratio to the extraction medium culture medium, place it horizontally and shake it,120rpm，37Extraction under ℃ conditions24hAfter the extraction is completed, invert and mix up10Next, take the extraction solution for testing. The culture medium used for extraction serves as a blank control. Pay attention to the prepared culture medium4Store at ℃ for no more than two weeks. The in vitro cytotoxicity test adoptsMTTMethod for testing: select those in good conditionL929Cells, digested and centrifuged, prepared into cell suspension, inoculated into96Hole culture plate. Multiple wells are set up in each group, and each well is inoculated100µLCell suspension, placed37℃、5%carbon dioxideCO2Incubation box cultivation24hDiscard the original culture medium and add100µLExtract solution and blank control solution, inCO2Continue to cultivate in the incubator.24hAfterwards, observe the cell morphology under a microscope. Discard the liquid in the well and add it to each well50µL1mg/mLAsepticMTTSolution（MTTuseMEMCulture medium configuration, ready to use, not suitable for culture medium preparation), continue cultivation2hDiscard the liquid in the hole and add100µLIsopropanol, shake and mix well, in570nmand650nmMeasure absorbance at wavelength and calculate cell survival rate.

Cytotoxicity testing is the use of in vitro cell culture methods to evaluate the potential cytotoxicity of biomaterials, medical devices, or extraction solutions. It is one of the important measurement indicators in the biological evaluation system of biomaterials and is also a necessary item for testing almost all types of medical devices and biomaterials. Cell toxicity testing can test the impact of biomaterials on cellular metabolic function in a short period of time, and can quickly screen whether materials have potential cytotoxicity.

Cell toxicity testing, as a means of detecting the toxicity of biomaterials, has the advantages of simplicity, high sensitivity, low cost, and short testing cycle. It is widely used as a biocompatibility testing and evaluation method for biomaterials and medical device products. The evaluation methods for the cytotoxicity of biomaterials have evolved from observing changes in cell morphology and quantity to assessing cell adhesion, proliferation, metabolism, and other aspects. The number of viable cells and cell proliferation ability are used as evaluation methods for assessing the biocompatibility of biomaterials. Specific experiments include neutral red uptake test, clone formation testMTTCytotoxicity testing andXTTCell toxicity tests and other methods, among whichMTTandXTTThe cytotoxicity test method is more widely accepted and applied due to its ability to quantitatively measure the cytotoxicity of materials.

MTTandXTTThe detection principle of cytotoxicity assay is that succinate dehydrogenase in mitochondria of living cells can enhance exogenous activityMTTReduced to water-insoluble blue purple crystalline formamide(Formazan)And deposited in cells, dead cells do not have this function. Dimethyl sulfoxide(DMSO)Or isopropanol or phenazine methyl sulfate(PMS)Can dissolve the formamide crystals in cells and use an enzyme-linked immunosorbent assay (ELISA) reader to detect them570nmMeasure the light absorption value at the wavelength, and within a certain range of cell numbers, the amount of crystal formation is proportional to the number of cells. According to the measured absorbance value(ODvalue)To determine the number of live cells,ODThe larger the value, the stronger the cell activity and the lower the toxicity. By calculating the proliferation rate of cells, the cytotoxicity of medical materials and their products can be quantitatively graded and evaluated. The survival rate of cells can also be calculated by diluting the extraction solution at different concentrationsIC50(Cell activity inhibition50%The concentration)Evaluate the cytotoxicity of biomaterials and their products.

Positive control substance for in vitro cytotoxicity testIt has the characteristics of good batch reproducibility, stable performance, and rich specifications, and can be used as a biological evaluation tool for medical devices-Ideal material for cytotoxicity testing.