[Optimization of isolation and culture of Lin- Sca-1+ cardiac stem cells from newborn mice]

Abstract

Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin^- (lineage-negative) Sca-1⁺ (stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0-3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1⁺ cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of Lin^- Sca-1⁺ cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of Lin^- Sca-1⁺ CSCs grew into spheres on the 5^th day, and over the whole bottom of the dish on the 7^th day. The growth curve showed that the cells were in logarithmic growth phase on the 3^rd day; (3) Immunofluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1⁺ cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of Lin^- Sca-1⁺ CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1⁺ CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by Lin^- Sca-1⁺ CSCs transplantation.