Cytoplasmic hybrid (cybrid) cell lines as a practical model for mitochondriopathies

Abstract

Cytoplasmic hybrid (cybrid) cell lines can incorporate human subject mitochondria and perpetuate its mitochondrial DNA (mtDNA)-encoded components. Since the nuclear background of different cybrid lines can be kept constant, this technique allows investigators to study the influence of mtDNA on cell function. Prior use of cybrids has elucidated the contribution of mtDNA to a variety of biochemical parameters, including electron transport chain activities, bioenergetic fluxes, and free radical production. While the interpretation of data generated from cybrid cell lines has technical limitations, cybrids have contributed valuable insight into the relationship between mtDNA and phenotype alterations. This review discusses the creation of the cybrid technique and subsequent data obtained from cybrid applications.

An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis

“…Remarkably, pyruvate even enables the proliferation of cells that have deleterious mutations in their mitochondrial DNA or lack it altogether (143B ρ 0 cells) (King and Attardi, 1989). Pyruvate has been hypothesized to act as a biosynthetic substrate or to maintain the cellular redox state in cells with ETC dysfunction via reduction by lactate dehydrogenase, which helps regenerate the NAD + that is lost upon ETC inhibition (Harris, 1980; Wilkins et al, 2014). The NAD + made through pyruvate reduction should facilitate glycolytic flux and thus ATP production in cells lacking ETC function, but the key metabolic consequence of pyruvate addition that allows such cells to proliferate is unclear.…”

Development of Treatments and Therapies to Target Mitochondrial Dysfunction

“…Initial attempts to recreate heteroplasmy in vitro led to the production of a cytoplasmic hybrid -known as a cybrid, created by fusion of a rho-zero (lacking mtDNA) transformed cell line with an enucleated cell line carrying donor mtDNA(s) [ 142 ]. Creation of cybrids carrying varying point mutations, deletions, and haplotypes have since been created to address a range of questions surrounding mitochondrial disease and the role of mtDNA in other diseases such as Parkinson's or diabetes [ 143 ].…”

The Potential Application of Mitochondrial Medicine in Toxicologic Poisoning

“…The next step involves dividing the existing cells that will lead to dilution of the existing mtDNA. This will eventually create a population of cells that lack mtDNA, now referred to as Bρ0.^Table 1 provides a list of currently reported ρ0 cell lines [6].…”

“…Techniques include measurement of oxygen consumption, cytochrome c oxidase activity, PCR of mtDNA, and southern blotting. All of the following techniques rely on demonstration of a negative result so will vary depending on the sensitivity of the test being used [6]. Another technique used to verify ρ0 status is testing for uridine/pyruvate auxotrophy.…”