YY/T 1876-2023Tissue engineered medical products - Quantification of remnant DNA in animal-derived biomaterials - Fluorescent staining method (English PDF)

1 Scope

YY/T 1876 specifies how to measure the DNA left behind in an animal-derived biomaterial, using a fluorescent staining method. Decellularised tissue, collagen, pericardium and bone matrix taken from animals are made safe by stripping out the donor cells, and residual DNA is the accepted indicator of how completely that was done: too much of it and the graft risks an immune reaction in the recipient. The document fixes how the sample is prepared, how the stain is applied and how the reading is converted into a residual DNA figure, so that results from different laboratories can be compared and a release specification can be enforced. For a manufacturer of xenogeneic implants or tissue-engineered scaffolds, this is the assay a Chinese reviewer expects the decellularisation validation and the batch release data to be based on.

This document specifies a method for the determination of remnant DNA in biological materials utilizing animal tissues and their derivatives. This document applies to final products or intermediate products of biological materials utilizing animal tissues and their derivatives, scaffold materials utilizing animal tissues and their derivatives used as matrices or scaffolds for tissue engineered medical products, and can also be used for human-derived decellularized matrix materials.

2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

YY/T 0771.1, Medical devices utilizing animal tissues and their derivatives - Part

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1 extracellular matrix; ECM Produced by cells and secreted into the extracellular space within tissues, it includes a homogeneous matrix (proteoglycans and glycoproteins) and filamentous collagen fibers. It has the function of connecting and supporting cells and is the basic framework

4 Test principle

This document designs a three-step method for the quantitative detection of remnant DNA in biological materials utilizing animal tissues and their derivatives, namely, proteinase K digestion and/or tissue homogenization of solid biological materials, DNA purification, and DNA detection by fluorescent staining. In addition to the test sample, a spiked recovery test sample must be set up simultaneously (i.e., a certain amount of DNA control substance is added to the test sample). In general, nucleic acid extraction usually uses one or more of the following methods. cell lysis, tissue homogenization, enzymatic hydrolysis and ultrasonication. In order to obtain ideal purified DNA, it is recommended to use a combination of enzymatic hydrolysis and/or tissue homogenization. This combined method is particularly suitable for situations where decellularized ECM materials are difficult to digest. In order to avoid the interference of digested decellularized ECM components on DNA detection, use a nucleic acid extraction kit to purify DNA. In this document, nucleic acid dyes (such as Picogreen fluorescent dye) are used for the quantitative detection of double- stranded DNA.

5 Samples, reagents, and instruments

5.1 Test sample Sample requirements are as follows:

a) Test sample. Decellularized matrix materials (final products or semi-finished products) and raw materials shall be sterilized and virus inactivated in accordance with YY/T 0771.1 and YY/T 0771.3 to ensure biosafety;

b) DNA standard curve samples. Use animal cell-derived DNA control samples to establish a standard curve;

c) DNA purification spike recovery sample. Add a certain amount of DNA control substance to another sample with the same amount as the sample to be tested, and use it to determine the spike recovery rate;

d) Negative control sample. Composed of phosphate-buffered saline (PBS) and bovine serum albumin (BSA), used to exclude contamination during DNA extraction. Note

1.For samples whose final products are not easily digested by enzymes (proteinase K, trypsin, etc.) due to the use of chemical cross-linking and other

5.3.2 Black 96-well plate.

5.3.3 Fluorescence microplate reader.

5.3.4 Low-adhesion DNase-free sterile centrifuge tube.

5.3.5 Low-retention sterile pipette tip.

5.3.6 Refrigerated centrifuge.

5.3.7 Vacuum dryer.

5.3.8 Oscillator.

5.3.9 Vortex mixer.

7 Result calculation

7.1 Data processing First, measure the standard and the sample to be tested 5 times to obtain the relative fluorescence intensity value; take the average of the relative fluorescence intensity (RFI) values of the 5 measurements; subtract the background (the RFI value measured when only TE is present and no DNA is present).