Abstract 11948: A Standardized Method to Isolate and Purify Cardiomyocytes from Individual Mouse Hearts Irrespective of Postnatal Age
Abstract only Rational: Primary cardiomyocytes (CMs) are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified CMs at any postnatal age, without the need for different age-depend...
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Published in | Circulation (New York, N.Y.) Vol. 144; no. Suppl_1 |
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Main Authors | , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
16.11.2021
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Online Access | Get full text |
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Summary: | Abstract only
Rational:
Primary cardiomyocytes (CMs) are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified CMs at any postnatal age, without the need for different age-dependent isolation procedures and cell culture, is lacking.
Objective:
To develop a standardized method that allows rapid isolation and purification of CMs in high yield and viability from individual neonatal, infant, and adult mice.
Methods and Results:
Using a novel
in situ
aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates [postnatal day 0-2 (P0-2)], hearts of C57BL/6J mice were subjected to Langendorff retrograde perfusion and enzymatic digestion. After subsequent tissue disaggregation and filtration, CMs were isolated in high yield (1.55-2.2 x 10
6
CMs/heart) and viability (~78%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal CMs, compared to non-myocytes, allowed their further enrichment by differential centrifugation, before purification at the bench by bead-based immunomagnetic cell separation. This involved depletion of endothelial cells (for infant and adult preparations) or non-myocytes (neonatal preparations). Together, these procedures resulted in highly purified CMs (~95%) from hearts of all ages within 1 hour. Moreover,
in situ
fixation immediately after tissue digestion via coronary perfusion, preserved the cytoarchitecture of isolated CMs (~94% rod-shaped CMs at all postnatal ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of CM area and nucleation state. RNA-sequencing of CMs purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters rather than by sexes; a finding consistent with cardiac size and shape being indistinguishable between P2 male (n=6) and female (n=6) hearts as determined by micro-computed tomography (X-ray 3D imaging).
Conclusion:
The procedures developed here provide a universal protocol for the rapid purification of high-quality CMs from individual hearts at any postnatal age, even those of neonates. |
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ISSN: | 0009-7322 1524-4539 |
DOI: | 10.1161/circ.144.suppl_1.11948 |