Establishment And Validation Of The Detection Method For Cell Cross-contamination Based On Microhaplotypes

Cell cross contamination refers to the contamination caused by the mixing of nontarget cells from within or outside species in the process of cell separation,culture and use.This problem has a long history,which can be traced back to the establishment of He La cell line,and has since increased in intensity,spreading to other cell lines and other species.If cross contaminated or incorrectly identified cell lines are used in research or production,it will lead to serious consequences,such as incorrect findings,unrepeatable experimental results,and safety risks for cell therapy products.Cell cross contamination detection methods include morphological analysis,isozyme analysis,chromosome karyotype analysis,human leukocyte antigen(HLA)typing,PCR species identification,single nucleotide polymorphism(SNP),short tandem repeat(STR)typing,etc.However,these methods suffer from a lack of sensitivity and specificity and limited applicability.The STR technique is widely used for the identification of humanderived cell lines,but has the disadvantage of a high mutation rate and insufficient ability to detect mixtures.In conclusion,there is no single method to ensure error-free cell cross-contamination detection,so it is important to establish a specific,sensitive and reliable system to detect cross contamination of cell lines.Microhaplotype(MH)are multi-allelic molecular markers consisted of two or more closely linked single nucleotide polymorphic loci within a given DNA segment.They are highly polymorphic and have a very low mutation frequency,which gives them a unique advantage in individual identification and hybrid DNA analysis.In this study,a method was developed for the detection of cross contamination of cell lines based on the microhaplotype principle.The process included simulated crosscontamination detection,STR typing validation,and validation of the applicability of combined karyotype analysis and isozyme analysis.This paper is intended to provide new tools for the establishment of quality control systems for cells used for research and therapeutic purposes and their related products.1.Establishment of cell culture system for simulating cell cross contaminationIn this study,human embryonic liver diploid cells(CCC-HEL-1),human lung fibroblast like cells(HLF),human skin fibroblasts cells(HSF),and human liver cancer cells(Hu H-7)were used to simulate cross contamination.The above cells were purchased from authoritative cell banks(authenticated)to ensure their authenticity.After the process of cell freezing,cell recovery and passaging,the corresponding culture system was established and the cells were expanded and cultured,laying the foundation for the subsequent simulated cross contamination assay.Each cell was treated separately in the operation to avoid cross contamination of cells during the culture process itself.2.Screening and verification of microhaplotype lociIn order to screen for microhaplotype loci with a high degree of polymorphism,in conjunction with the objectives of this experimental study,we developed the following four criteria:(1)Give priority to the sites for validation analysis in Asian populations.(2)The greater the number of effective alleles(Ae),the stronger the ability to detect the mixture of microhaplotypes,selecting loci with Ae greater than 3.(3)To avoid linkage disequilibrium,loci with MH intervals greater than 10 Mb on autosomes and greater than 5 Mb on sex chromosomes can be selected.(4)The more alleles contained in the microhaplotype,the lower the probability of random matching and the better the discrimination ability,giving preference to microhaplotype loci containing triplet/quadruplet SNPs or loci with a higher number of haplotypes.A total of 131 microhaplotype loci were obtained by optimal screening of the microhaplotype detection population,Ae values,chromosome locus spacing and SNP polymorphisms.As an important indicator for evaluating microhaplotypes,Ae value can well reflect the ability of microhaplotype-based methods to detect mixtures of DNA from different sources.Therefore,a batch of 36 loci with an average Ae=4.2 was selected and validated by PCR amplification and high-throughput sequencing analysis using CCC-HEL-1 and Hu H-7 cells as samples.The results showed that the screened loci had high polymorphism and could be used for subsequent analysis.3.Establishment of cell cross-contamination detection method based on microhaplotypeThe study used 131 optimally screened loci to construct a microhaplotype assay system with HEL,HSF,HLF and Hu H-7 cell lines to simulate cell cross-contamination.The 17 cell detection samples were divided into single cell group(HEL,HSF,HLF and Hu H-7)and simulated cell cross contamination group,where four mixing ratios(1:9,1:19,1:49 and 1:99)were set for different cell combinations in the simulated contamination group to explore the detection sensitivity of the constructed microhaplotype system.By extracting the DNA of different combinations of simulated contaminated cells,establishing a multiplex PCR amplification system,and performing high-throughput sequencing,it could detect 1% of cell cross contamination.The microhaplotype assay system composed of 131 selected loci has the advantages of sensitivity,specificity,stability and reliability.4.Validation of cell cross-contamination detection method based on microhaplotypeTo verify the validity of the microhaplotype assay system developed in this study,firstly,single cell samples and simulated cross-contaminated cell samples with different mixing ratios(1:3 and 1:9)were selected for comparison with the STR typing method.The results showed that the microhaplotype analysis were consistent with those of the STR assay,and the sensitivity of the microhaplotype assay was much higher than that of the STR.When cross-contamination of less than 10% is encountered,the STR method is unable to distinguish between stutter peaks and cell cross-contamination.In contrast,the microhaplotype method detect no stutter pseudo-peaks and at least 1%cross-contamination can be detected.In addition,in order to verify the applicability of the microhaplotype system,especially for the detection of cross contamination of cells between different species such as human and mouse,a combination of "Microhaplotype analysis + Isozyme analysis + Chromosome karyotype analysis" was used in simulation detection.The results showed that the combination was able to identify cytogenetic stability and cross-contamination of cell lines of different generic origin.In this study,microhaplotype genetic markers are used for the identification of cell cross contamination for the first time.And a new cell cross contamination detection method is successfully established,making up for the deficiency of STR identification.This is of great importance to ensure the reliability and accuracy of cell research results,and also provide a new option for quality control of cell preparations.