Table 2.

Summary of the applications of in vitro 2D and 3D cell culture methods for MM research and their main advantages and disadvantages.

Method	Application to MM research	Advantages	Disadvantages
2D cell culture	*Large scale drug testing *Identification and/or validation of novel biomarkers. *Investigating the role of genes in MM progression.	*Cost-effective *Easy handling. *Easy to maintain. *High throughput capacity.	*Drug sensitivity data generated from this method does not always reflect that of the in vivo/clinical counterpart. * Lack of 3D structure; limited cell-cell interactions; unnatural substrate. *Lack of cellular heterogeneity/complexity compared to the original tumour. * Gene expression less similar to in vivo tumours.
3D cell culture (includes spheroids, TFS and organ-on-a-chip)	*Studying therapeutic efficacy of novel drugs. *Identification and/or validation of novel biomarkers. *Studying cell-to-cell and cell-to-extracellular matrix signaling.	*More representative of the in vivo tumour structure/complexity. *Gene expression more similar to in vivo tumours. * Drug response better reflects in vivo/clinical drug response. *Increased cell-to-cell and cell-to-extracellular matrix signalling.	*TFS and organ-on-a-chip require access to fresh surgical MM tumour samples = low throughput capacity. *Complex handling. *Less cost-effective.
Whole genome sequencing	*Studying all types of MM-specific genetic variation across the entire genome.	*Detects coding, non-coding and structural variants across the entire genome.	*High associated cost. *Large volume of data to process and store. *Numerous variants of unknown significance can be detected. I.e. limited knowledge to fully understand / appreciate the significance of detected unknown variants.
Transcriptome sequencing	*Studying all types of aberrant MM-specific mRNA / transcript variation.	*Rapid, precise, quantitative measurement of gene expression. *High sensitivity enables detection of low-abundance transcripts. *DNA sequences can be unambiguously mapped to unique regions of the genome instead of relying on existing genome sequence data. *Useful for the discovery of single-nucleotide polymorphisms and rare mutations. *More affordable compared to whole genome sequencing.	*Transcript quantitation can be affected by biases introduced during cDNA library construction and sequence alignment. *Accurate sequence annotation and data interpretation can be computationally challenging in the absence of pre-existing reference genome(s).
Targeted sequencing	*Studying unique MM-specific alterations at the sites of specific regions of the genome (i.e. exosomes) or subset of genes.	*Significantly less time-consuming and more cost-effective than whole genome sequencing. *Specific areas of the genome can be sequenced at a greater depth than whole genome sequencing. *Reduced volume of data to process and store than whole genome sequencing.	*Only focuses on limited regions of the genome, meaning it does not take into account any other genetic variants outside of the focus/target gene panel.
Droplet digital PCR	*Studying unique MM-specific gene copy number variations, DNA mutations or deletions. *Detection and validation of MM-specific biomarkers.	*Provides an absolute and independent quantification of DNA without the need for a standard curve. *Generated data is more accurate and reproducible than conventional qPCR. *Capable of detecting very low concentrated target molecules from variably contaminated samples.	*Equipment and reaction running costs are more expensive than conventional qPCR. *Requires advanced skill and handling compared to conventional qPCR.