Microwell respirometry

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Microwell respirometry

Oroboros comments on microplate respirometry

In respirometry, high throughput is not equivalent to high output.
» O2k specifications


Respirometry with cryopreserved blood cells

Successful O2k application

O2k high-resolution respirometry has been successfully applied on cryopreserved cells from human blood samples to detect alterations reflecting human systemic physiological disease. Using blood samples for OXPHOS analysis extends the scope of diagnostic applications of the O2k, when compared to muscle biopsies. Respiration of blood cells is measured with cells in suspension, using the O2k technology. This reduces stress conditions for the cells compared to cells seeded on microplates.

Reports on this successful application of cryopreserved blood cells with O2k high-resolution respirometry can be found by Karabatsiakis and colleagues (2014) [1] which are based on

(1) functional assays with blood cells in suspension;
(2) immediate measurement in the OROBOROS O2k after thawing upon cryopreservation.

Highlights of these recent developments:

» O2k-Publications: Cryopreservation

Blood cells stressed in 96-well plate

  • In contrast, Keane et al (2015) [2] found mitochondrial injuries after cryopreservation of blood cells. The authors state: The Seahorse XFe96 Flux analyzer was used according to manufacturer's instructions. .. In brief, cells were seeded into 96-well plates at a density of 3.5 x 105 cells/well and allowed to adhere overnight. .. then incubated for 60 min at 37 °C in an non-CO2 incubator, to allow the cells to become equilibrated with the new medium. The reductions in ROUTINE respiration, ET-capacity and coupling after 1 month cryopreservation may be related to
(1) measurement on blood cells adherent in microwells;
(2) delayed measurement following overnight plus 60 min stress condition.

References

  1. Karabatsiakis A, Bock C, Salinas-Manrique J, Kolassa S, Calzia E, Dietrich DE, Kolassa I-T (2014) Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression. Translational Psychiatry 4:e397. »Bioblast link«
  2. Keane KN, Calton EK, Cruzat VF, Soares MJ, Newsholme P (2015) The impact of cryopreservation on human peripheral blood leukocyte bioenergetics. Clin Sci (Lond) 128:723–33. »Bioblast link«


Are microwell cell cultures physiological?

Biologists go to great lengths to figure out just the right cocktail of chemicals on which to propagate the cells they want to study. Not too much nitrogen—just the right amount of sulfur—and so on. But then the whole mix is thrown into a flat-bottomed microwell where the newly sprouted cells bunch up and do not exactly look or behave like cells grown au naturel. Hoping to give lab-cultured cells the chance to more closely resemble their in vivo brethren, materials science researchers have designed a technique for growing cells in three dimensions on top of a cushion of air.